Pharmaceutical preparation for treating blood coagulation disorders

There is disclosed a pharmaceutical preparation for treating blood coagulation disorders which comprises purified prothrombinase factors, in particular purified prothrombin and optionally purified factor Xa as active component.

The present invention relates to a pharmaceutical preparation, in 
particular for the treatment of blood clotting disorders, comprising 
coagulation factors which are components of a prothrombinase or of a 
pro-prothrombinase, respectively. 
Prothrombinase is an enzyme substrate complex which forms on a phospholipid 
surface and enables the activation of prothrombin. By definition, 
prothrombinase consists of factor II (prothrombin), activated factor X 
(factor Xa), cofactor V or Va, respectively, phospholipids and calcium 
ions. In vivo, these factors are present as a transient complex for the 
activation of prothrombin and the formation of thrombin. 
A corresponding pro-prothrombinase is defined as a complex of factors which 
are present at least partially modified or activated for the formation of 
a prothrombinase. Pro-prothrombinase thus is to be understood as a proform 
of a prothrombinase and as complex in which one or more components are 
present in their pre-stages, as zymogens or as proforms and which is 
formed on the basis of affinities of the components to one another. 
Hemophilia A arises from an X-chromosomal recessive hereditary factor VIII 
deficiency and becomes manifest by severe blood coagulation disorders. For 
controlling acute bleedings, coagulation-active plasma protein 
concentrates are used in most cases, primarily factor VIII concentrates. 
However, in the classical treatment of hemophilia A-patients with factor 
VIII preparations, antibodies to factor VIII will form in about 20% of the 
cases, which antibodies lead to an inhibition of the factor VIII 
administered with these preparations. One then says that a patient has 
formed a functional inhibitor to factor VIII and has developed a so-called 
factor VIII-inhibitor hemophilia or acquired hemophilia. 
At present, several methods are used for the therapy of hemophilia 
A-patients having a factor VIII inhibitor hemophilia: 
1.) Treatment with high doses of a factor VIII preparation: 
With this, the antibody directed against factor VIII is neutralised in 
vivo, and the excess of factor VIII can unfold its hemostatic cofactor 
activity. By repeated administration over an extended period of time, the 
afflicted patient is desensitized against factor VIII and subsequently in 
many cases may be subjected to the common factor VIII concentrate therapy. 
This mode of procedure requires extremely large amounts of factor VIII, is 
time-consuming and may involve massive anaphylactic side effects at the 
onset of treatment. 
2.) Treatment of factor VIII inhibitor patients with immunoglobulin 
preparations which contain antiidiotypic factor VIII antibodies: 
This route of therapy at present is subject to intensive research. However, 
no final judgment is as yet possible on the efficacy of such a treatment. 
3.) Immunoadsorption: 
A further complex method for removing factor VIII inhibitors is the 
extracorporeal immunoadsorption either on lectins which bind 
immunoglobulins (protein A, protein G), or on immobilized factor VIII, to 
which the antibody formed against factor VIII is bound. This method is 
complicated for the patient because in this case he is bound to an 
apheresis machine, as with the previous methods it may not lead to a 
cessation of an acute bleeding and, furthermore, it is expensive. 
4.) APCC and derivatives 
At present, the therapy of choice is the administration of activated 
prothrombin complex concentrates (APCC), FEIBA.RTM., AUTOPLEX.RTM., which 
can be utilized to stop acute bleedings also in patients having high 
inhibitor titers (cf., e.g., DE-PS 31 27 318 (2)). 
Based on the activated prothrombin complex factor concentrates, a component 
which is also contained in the former, i.e. activated factor VIIa, has 
been suggested as a therapeutic principle for factor VIII inhibitor 
patients via the extrinsic coagulation pathway. A corresponding 
preparation, i.e. recombinant factor VIIa, at present is being clinically 
tested (Hedner et al., Transfusion Medicine Reviews 7 (2): 78-83 (1993)). 
Preclinical trials, e.g. on dogs suffering from hemophilia A, have, 
however, indicated that the treatment with recombinant factor VIIa is 
ineffective. Similarly, also the success rate in human application is 
varying. A further disadvantage of recombinant factor VIIa consists in 
that on account of its very short half-life in vivo, the latter must be 
administered frequently in high doses per day to control severe bleedings, 
if possible at all. In such instances it is attempted to administer factor 
VIIa together with antifibrinolytic agents so as to aid its effect. 
In the literature (e.g. DE 44 16 180 A1) it has also been proposed to 
therapeutically utilize a combination of factor Xa and phospholipids for 
the treatment of hemophilia A inhibitor patients. In vivo tests on factor 
VIII-deficient dogs with inhibitor showed that in a suitable dosage such a 
combination is able to stop an acute bleeding. The therapeutic spectrum of 
such a preparation is, however, comparatively narrow, since the effective 
and the thrombogenic dose are closely adjacent, which could, e.g., be 
demonstrated in rabbits in the Wessler model, since in particular 
phospholipids constitute an increased thrombogenicity risk. 
The present invention thus has as its object to avoid the disadvantages of 
the methods described and to provide a therapy principle for the treatment 
of blood clotting disorders, in particular for the treatment of factor 
VIII inhibitor patients, which enables, i.a., a simple administration, an 
effective onset of action, an increased half-life and the avoiding of 
thrombogenic side effects. 
According to the invention, this object is achieved with a pharmaceutical 
preparation for the treatment of blood coagulation disorders comprising, 
as active components, at least two coagulation factors which are 
components of a prothrombinase or of a pro-prothrombinase, respectively, 
in particular with a preparation comprising, as active components, 
purified prothrombin and purified factor Xa, in the solubilized state, one 
of the factors besides prothrombin preferably being activated. Preferably, 
the components are purified at least so far as to be free from endogenous, 
i.e. starting-material-derived, phospholipids, but also in particular 
phospholipid vesicles. 
Thus, on the one hand, a premature thrombin formation is prevented and the 
stability of the pharmaceutical preparation is ensured, and, on the other 
hand, the risk of thromboembolic side effects is minimized. 
According to the invention, a mixture or a complex of at least two 
components of the prothrombinase is understood as a "partial 
prothrombinase". 
Beside the components of the prothrombinase or of the pro-prothrombinase, 
respectively, advantageously further factors of blood coagulation and 
fibrinolysis are contained so as to obtain an attenuated effect, in 
particular an increase, decrease, acceleration or deceleration of 
hemostasis. Accordingly, activators or pro-activators of blood 
coagulation, among them factors of intrinsic or extrinsic blood 
coagulation, can further be contained as zymogens or as activated factors, 
as well as their agonists or antagonists or inhibitors. Besides, also the 
corresponding combinations are possible as preparations which are 
administered separately. Among them are the combination with fibrinogen 
which above all is suitable for local application. 
According to a preferred embodiment, however, the pharmaceutical 
preparation substantially consists of the "partial prothrombinase", the 
components of prothrombinase or of pro-prothrombinase, respectively, 
preferably as a complex. This complex can be purified and treated in a 
simple manner, in particular treated chemically and/or physically to 
inactivate molecular, microbial or viral pathogens. 
The factors of the pharmaceutical preparations according to the invention 
are contained in a form which enables activation of at least one factor, 
or in which at least one factor has already been activated. As factors, 
preferably human factors are used. The factors are contained in the 
pharmaceutical preparations according to the invention, which are 
preferably selected from the group consisting of the factors II, V, Va, X 
and Xa. 
As inventive preparation of a partial prothrombinase or pro-prothrombinase, 
respectively, preferably combinations of factors II and V or Va, 
respectively, as well as X and V or Va, respectively, are provided. There, 
it is particularly preferred that the preparation according to the 
invention substantially consists of these combinations. Likewise, a 
pro-prothrombinase comprised of factors II and X, optionally in 
combination with factor V or Va, respectively, is a preferred embodiment 
of the present invention. 
Therein, native factors can be used, e.g. proteins or their equivalents 
recovered from plasma or from a plasma fraction, which are, e.g., encoded 
by recombinant nucleic acids. Furthermore, also respective derivatives 
which comprise the modified proteins or fragments are also suitable as 
long as they are activatable or have the respective activity to modulate 
the generation of thrombin. 
The preparation according to the invention has the advantage that, despite 
its high stability in vitro, it is also stable in vivo until its efficacy 
is shown by the activation of prothrombin and the generation of thrombin 
at the site of the wound or of the bleeding. By the contact with the 
cellular components of the vascular system, e.g. blood cells and vessel 
walls, in particular phospholipid-containing surfaces, thrombin is 
generated in situ and hemostasis is promoted. On account of the local 
efficacy, systemic side effects, such as, e.g., thromboembolic 
complications, are avoided. 
To effect a controlled influence on hemostasis, it is preferred to use 
highly purified factors which have been purified from interfering 
contaminants, in particular from a thrombin activity. The factors are 
especially suitable which have been purified by chromatographic methods, 
such as ion exchange chromatography, hydrophobic chromatography, affinity 
chromatography and/or molecular exclusion chromatography. Thereby, 
specific activities of at least 50% of the theoretic purity, in particular 
at least 70%, preferably at least 90% up to theoretic purity can be 
attained for the individual factor. Accordingly it is also preferred to 
use factors which are substantially free from and devoid of denaturing 
products and thus are present as purified active factors, enzymes or as 
activatable zymogens, respectively. 
Furthermore, it is preferred to carry out a treatment for the inactivation 
of infectious pathogens, e.g. by a treatment with chemicals and/or by a 
physical treatment, such as a heat treatment, radiation or filtration, in 
particular nanofiltration. According to a preferred variant, the factors 
of the pharmaceutical preparation according to the invention are treated 
with detergents, which, on the one hand, leads to the inactivation of 
viruses and, on the other hand, solubilizes any phospholipids possibly 
present. 
Phospholipids may be contained in preparations of blood coagulation 
factors, e.g. from plasma or from a plasma fraction or from a cell 
culture, respectively. The special treatment of the factors for separation 
of the naturally present phospholipids comprises the solubilisation 
thereof, on the one hand, and the separation of the phospolipids by the 
purification methods mentioned above, on the other hand. 
Although the preparation according to an embodiment may be utilized in 
combination with exogenic phospholipids, the preparations according to the 
invention are preferably free from added phospholipids and contain less 
than 0.01 mg of phospholipids/U prothrombin. Due to the possible 
thrombogenic effect of phospholipids, the thrombogenicity risk is further 
markedly reduced. According to a particularly preferred embodiment, the 
preparations are free from detectable phospholipid. 
According to a further embodiment, the preparation according to the 
invention furthermore contains magnesium ions. These ions act 
competitively to calcium ions and can displace the calcium ions in the 
prothrombinase or pro-prothrombinase, respectively. Thus, a premature 
thrombin formation in a solution of the preparation according to the 
invention is prevented and the preparation thus is stabilized to an extent 
that it remains stable in a solution even for hours. 
It has been shown that the pharmaceutical preparation may even be provided 
as a stable infusion solution, primarily if it is ensured that it does not 
contain any free calcium ions. For complexing the calcium ions, also a 
content of a pharmaceutically acceptable chelating agent, e.g. EDTA, and 
related structures, such as citrate, are suitable. 
The preparation according to the invention comprises a biological efficacy 
in animal models which is comparable to FEIBA.RTM. and it can markedly 
reduce the coagulation time of a factor VIII inhibitor plasma. It can 
completely normalize the prolonged bleeding time and intensity of bleeding 
of factor VIII inhibitor rabbits and of von Willebrand factor inhibitor 
rabbits. By providing purified blood coagulation factors, e.g. purified 
prothrombin and purified factor Xa, the toxicity of the preparation 
according to the invention is clearly reduced as compared to FEIBA.RTM.. 
Thus, e.g., the effective combination of factor Xa and prothrombin proved 
negative in the Wessler-thrombosis model (J. Appl. Phys. 14 (1959), 
943-946), i.e. for this combination, no thrombogenic effects could be 
detected in the Wessler model even at higher doses as compared to 
activated prothrombin complex. 
In this thrombogenicity model, rabbits are anesthesized with pentobarbital, 
whereupon, under an additional local anesthesia, the vena jugularis is 
prepared and provided with loose ligatures at a distance of 2 cm. Finally, 
the substance to be tested is injected within 15 seconds into the ear vein 
opposite the vena jugularis. After further 25 seconds, the ligatures are 
closed, and after a waiting time of 10 min the ligatured vein section can 
be withdrawn and dissected in a Petri dish filled with citrated buffer and 
evaluated. The evaluation criteria, modified according to Wessler, are as 
follows: no formation of thrombi=0, a few small thrombi=0.5-1, a few, 
medium-sized and many small thrombi=2, many medium-sized thrombi=3, a few 
large thrombi=3.5, one coherent thrombus=4. 
The components of the pharmaceutical preparation according to the invention 
preferably have been purified to such a purity that even at a dose of at 
least 150 U prothrombin/kg it is free from thromboembolic side effects, 
expressed by a score in the Wessler thrombosis model of 3 at the most, 
preferably 2 at the most, in particular less than 2. 
In the combination preparation of the invention, prothrombin is contained 
preferably at a specific activity of at least 5 U/mg protein, more 
preferred at least 6, in particular at least 7, corresponding to 50, 60 or 
70% of the theoretic purity. The factor X or Xa preparation used should 
preferably have a specific activity of at least 100 U/mg protein, factor 
Xa preferably being mainly contained as factor Xa.beta.. Factor V or Va, 
respectively, is used as co-factor at an approximately equimolar ratio to 
the coagulation factor of the partial prothrombinase or 
pro-prothrombinase, respectively. Preferably, the ratio is (0.01-2):1 
(mol/mol), most preferably (0.5-2):1. 
The preparation used should be as free from thrombin as possible, and it 
should be possible to detect this freedom from thrombin by suitable, 
preferably chromogenic, tests (e.g. with the chromogenic substrate TH-1 
from IMMUNO AG). 
It has been shown that, if the coagulation factors, such as, e.g., 
prothrombin and factor Xa, are present as a complex in the preparation 
according to the invention, the preparation has an increased stability as 
compared to conventional preparations, and the complex furthermore can be 
subjected to a further treatment for purification and/or inactivation of 
viruses. In a preferred embodiment of the preparation according to the 
invention, the preparation further comprises antithrombin III in 
stabilizing amounts, optionally together with heparin. It is possible to 
demonstrate also such a preparation's freedom from thromboembolic side 
effects according to the Wessler test in the absence or also after 
unmasking of the heparin, i.e. neutralisation and/or separation of the 
heparin, in a sense that the score of 3 will not be reached. 
A complex consisting of at least two coagulation factors which are 
components of a prothrombinase or of a pro-prothrombinase, respectively, 
in particular in highly purified form, is of basic importance as a 
"partial prothrombinase complex". By the presence of calcium ions, such as 
calcium chloride, the activity of a preparation on the basis of this 
complex can be accelerated by a multiple. It has also been shown that a 
preparation comprising this complex and further calcium ions can be used 
for the preparation of a reagent for diagnostic purposes. A reagent which 
further comprises thrombin activity and optionally phospholipids is, e.g., 
suitable to determine factor V cofactor activity. A diagnostic method 
using this reagent with or without activated protein C, a proteolytic 
inactivator of factor V, furthermore enables estimation of an extent of an 
inactivation of factor V or a mutation-caused resistance to activated 
protein C. 
Surprisingly, within the scope of the present invention it has also been 
found that a pharmaceutical preparation comprising purified prothrombin as 
the only active component has an effect in vivo comparable to the 
combination preparation of the invention in the treatment of coagulation 
disorders, although in vitro with prothrombin alone no shortening of the 
coagulation time of a factor VIII inhibitor plasma could be achieved. 
Thus, a medical indication besides hereditary prothrombin deficiency for a 
pharmaceutical preparation comprising prothrombin as the only active 
component has for the first time been shown. 
The invention thus also relates to a pharmaceutical preparation, in 
particular for the treatment of coagulation disorders, comprising purified 
prothrombin as the only active component. 
Preferably, the prothrombin has been purified to such a degree that even at 
a dose of at least 150 U prothrombin/kg, it is free from thromboembolic 
side effects, expressed by a score of 3 at the most, preferably 2 at the 
most, in particular less than 2, in the Wessler thrombosis model. 
The combination preparation according to the invention and the prothrombin 
preparation, respectively, preferably comprise less than 0.1 U of factor 
VIII:C or of factor VIII:Ag/U of prothrombin, and less than 0.1 U of 
factor IX/U of prothrombin or less than 0.1 U of factor X/U of 
prothrombin, respectively. By this, the undesired formation of or reaction 
with antibodies to these proteins can efficiently be prevented, and the 
risk of side effects can be reduced. 
Although the prothrombin preparation can be used in combination with 
phospholipids, according to a further preferred variant of the 
preparations according to the invention, these are free from added 
phospholipids and contain less than 0.01 mg of phospholipids/U of 
prothrombin. According to a particularly preferred embodiment, the 
prothrombin preparations according to the invention are free from 
detectable phospholipid. 
The dosage of the preparations according to the invention is based on the 
dosage of the equivalent components in FEIBA.RTM.. The Factor Eight 
Inhibitor Bypassing Activity (FEIBA) is defined as that activity of such a 
preparation which reduces the coagulation time of a factor VIII inhibitor 
plasma to 50% of the blank value in a clotting test as described in AT 
350726. On account of the high purity of the components, the advantages of 
the preparation according to the invention as compared to FEIBA.RTM. 
reside in a reduced load of plasma proteins on the patient. In particular, 
the absence of factor VIII:Ag excludes the anaphylactic side effect. Thus 
it is possible to administer the preparations of the invention at a 
concentration which provides for a dose which, e.g., comprises at least 50 
U prothrombin/kg body weight, this dose for the first time for such 
preparations being administrable even in a bolus injection because of its 
freedom from side effects, and thus the otherwise time-consuming 
administration of such high doses as an infusion can be avoided. 
Usually the preparations according to the invention are administered at a 
dose of e.g. from 50 to 150 U prothrombin/kg body weight, the maximum 
doses, however, possibly lying far beyond these 150 U/kg body weight 
(e.g., up to 300 or up to 500, respectively) without possible 
thromboembolic side effects. 
Thus, the invention also relates to administration forms of the 
pharmaceutical preparations of the invention which comprise a dose of at 
least 50 U prothrombin/kg body weight, preferably of between 50 and 500 
U/kg body weight. 
These administration forms may be ampouls or syringes which are already 
provided for an immediate application, or similar directly or indirectly 
applicable forms. Among them are containers which are suitable for 
infusion, intramuscular or subcutaneous application or sets consisting of 
a container comprising the active agents as a lyophilisate and a container 
comprising a pharmaceutically acceptable solution suitable for 
reconstitution of the lyophilisate. Usually, the pharmaceutically 
acceptable solution or the pharmaceutical preparation, respectively, 
contains salts, preservatives, buffers and the like in an aqueous solution 
(cf. Remington's Pharmaceutical Sciences, 15th edition, Easton: Mack 
Publishing Co., pp. 1405-1412 and 1461-1487 (1975), and The National 
Formulary XIV., 14th edition, Washington: American Pharmaceutical 
Association (1975)). Examples of non-aqueous solutions are propylene 
glycol, polyethylene glycol, vegetable oils and injectable organic esters, 
such as ethyl oleate. Aqueous carriers are, e.g., water, optionally mixed 
with alcohol, salt solutions (NaCl), Ringer's dextrose etc.. As the 
preservatives, antimicrobial substances, antioxidants, chelating agents or 
inert gases may be used. 
The preparations according to the invention are also suitable for local 
treatment, application forms being chosen which become active at the site 
of a bleeding. Among them are solids or liquids, preferably in the form of 
a powder, an adhesive tape or a wound cover, respectively, ointments, 
suppositories, capsules, in particular gastric-juice-resistant capsules, 
but also drops or sprays. 
The coagulation factors used may be both of plasmatic origin and proteins 
prepared by recombinant DNA technology. What is essential in both 
instances is that they are present in the pharmaceutical preparation in 
purified form, in particular in a form freed from endogenous and exogenic 
phospholipids. Preferably, the pharmaceutical preparations according to 
the invention are provided in lyophilized form offering the known 
transportation, storage and application advantages. As the reconstitution 
solution, a pharmaceutically acceptable solution which optionally 
comprises ATIII or heparin, respectively, is suitable. Due to the high 
degree of purity of the components of the preparations according to the 
invention, they can be reconstituted to an optically clear solution after 
a short reconstitution time at room temperature, preferably less than 5 
min, in particular less than 1 min, with e.g. at least 10 U 
prothrombin/ml, concentrations of even up to 200 U prothrombin/ml solution 
being attainable. An optically clear solution is defined by a maximum of 
the absorbance at 600 nm of 0.1 (for a solution having a protein content 
of at least 5% by weight, at a layer thickness of 1 cm), based on the pure 
(buffer) solution as a reference. Alternatively, also a solution having 
less than 70 light scattering units (LSU), determined by measurement in 
the nephelometer at 340 nm and a layer thickness of 1 cm is considered to 
be clear. 
In contrast to the hitherto known preparations for treating blood clotting 
disorders, the preparations according to the invention are extremely 
stable, i.e. they can be allowed to stand for a longer period of time 
before being administered. For example, according to the product 
information, FEIBA.RTM. in its ready-to-administer state should not be 
left to stand for more than 1 hour, whereas the preparations according to 
the invention as a ready-to-use solution do not exhibit any clotting 
activation or thrombogenicity even for a period of 3 hours or more at room 
temperature, and thus the preparations according to the invention may also 
be provided as infusion solutions which can be administered over a period 
of several hours. For the same reasons it is also possible to administer 
the preparations of the invention over an extended period of time as 
infusion solutions. It has been shown, however, as regards the 
thromboembolic side effects, there is no substantial difference between a 
bolus injection and a slow infusion with the preparations according to the 
invention. 
According to a preferred embodiment of the present invention, the 
pharmaceutical preparations are made available in suitable application 
devices, preferably as a lyophilisate in syringes which allow for an in 
situ reconstitution with a pharmaceutically acceptable solution. For the 
combination preparation an application device is suitable which is shown 
in FIG. 1, wherein the lyophilisates of prothrombin and factor Xa, e.g., 
are preferably separately stored and can be administered when required 
after an in situ reconstitution by means of a double-chamber syringe. 
With the pure prothrombin preparation it suffices to provide the 
prothrombin in a simple syringe, preferably in the lyophilized state, 
optionally with a pharmaceutically acceptable solution for reconstitution 
(cf. FIG. 2). 
The prothrombin preparation may, however, also be provided as a liquid 
preparation or in liquid-deep frozen form, respectively. 
Particularly if they are recovered from plasmatic proteins or from cell 
cultures, the pharmaceutical preparations according to the present 
invention may be subjected to one or several virus inactivation treatments 
or treatments for virus depletion, respectively, e.g. to a chemical or a 
chemical-physical treatment, to a heat and/or a detergent treatment 
according to EP 0 159 311, EP 0 519 901 or EP 0 674 531, or to a physical 
treatment, such as nanofiltration. 
The preparations according to the invention anable a safe and simple 
treatment of blood coagulation disorders, in which an effective onset of 
action can be observed within a very short time. 
It has been shown that the effective onset of action occurs more rapidly 
when administering the complex of factor II and factor Xa than with a 
preparation containing factor II exclusively. To treat a bleeding 
complication in a patient suffering from inhibitor hemophilia it is, thus, 
advantageous to treat the bleeding initially by administering the factor 
II/Xa complex and thus attain a rapid hemostasis and to continue the 
therapy with maintenance doses of a preparation containing factor II only, 
to avoid further bleedings. 
Furthermore, the long half-life of the preparations according to the 
invention and their freedom from thrombogenic side effects or the absence 
of an anaphylactic reaction, which causes an increase in the inhibitor 
titer, enable a substantially improved treatment of the patient affected 
with a risk of blood coagulation disorders as compared to known methods. 
On account of the high concentration or dose of the preparation according 
to the invention, the patient may receive a depot of the active substance 
which reduces the need of frequent treatments. The patient may also remain 
without treatment for a longer period of several days and optionally may 
treat himself like an outpatient by self-injection, optionally 
subcutaneously. 
The invention further relates to a method of preparing a purified 
pharmaceutical prothrombin preparation which is characterized in that a 
prothrombin complex concentrate is subjected to chromatographic 
purification and the prothrombin-containing fraction is processed to a 
pharmaceutical preparation. With the method according to the invention a 
prothrombin preparation is provided which fully meets all the requirements 
in respect of purity for the pharmaceutical preparations of the invention, 
in particular as regards the freedom from side effects determined by means 
of the Wessler test. 
In particular, the method according to the invention is characterised by 
the combination of the following steps: 
providing a prothrombin complex preparation in the solid state or as a 
prothrombin-containing solution, 
virus inactivation treatment, preferably by heat treatment, in particular 
in the solid state, 
optionally dissolving the prothrombin complex preparation, thereby 
obtaining a prothrombin-containing solution, 
optionally treating the prothrombin-containing solution with an alkaline 
earth salt as the solid carrier, prothrombin being adsorbed and 
subsequently desorbed, 
optionally concentrating one or several times, preferably by precipitation 
or ultra/diafiltration, and gel filtration of the prothrombin-containing 
solution, 
treating the prothrombin-containing solution with an anion exchanger, the 
prothrombin being adsorbed and subsequently selectively desorbed, 
treating the prothrombin-containing solution with a hydrophobic 
chromatography material, and 
finishing the prothrombin-containing solution into a pharmaceutical 
preparation. 
Preferably, calcium phosphate, barium sulphate or aluminum hydroxide can be 
used as the alkaline earth salt. In principle, all anion exchangers which 
have an affinity to prothormbin can be used as anion exchangers, such as, 
e.g., DEAE-Sephacel.RTM., DEAE-Sephadex.RTM., DEAE-Sepharose CL6B.RTM., 
DEAE-Sepharose Fast Flow.RTM., QAE-Sephadex.RTM., Q-Sepharose Fast 
Flow.RTM., Q-Sepharose High Performance.RTM., Q-Sepharose Big Beads.RTM. 
(all from Pharmacia), 
DEAE-Tris-Acryl.RTM., DEAE-Spherodex.RTM., Q-Hyper-D.RTM. (all from 
Sepracor); 
Macroprep DEAE.RTM., Macroprep Q.RTM. (all from BioRad); 
DEAE-Toyopearl.RTM., QAE-Toyopearl.RTM., Toyopearl Super-Q.RTM. (all from 
Tosohaas), 
Protein PAK DEAE.RTM. (Waters); 
Fractogel EMD-TMAE.RTM., Fractogel EMD-DEAE.RTM., Fractogel EMD-DMAE.RTM., 
Licrospher 1000 TMAE.RTM., Licrospher 1000 DEAE.RTM. and Licrospher 4000 
DMAE.RTM. (all from MERCK). 
As the gel for the hydrophobic interaction chromatography, preferably 
Phenyl-Sepharose High Performance.RTM. (Pharmacia), yet also other 
chromatography gels, such as, e.g., Butyl-Sepharose.RTM., 
Octyl-Sepharose.RTM., Phenyl-Sepharose.RTM., Phenyl-Sepharose Fast Flow 
High Sub.RTM., Phenyl-Sepharose Fast Flow Low Sub.RTM. (all from 
Pharmacia), 
Fractogel TSK-Butyl.RTM. (MERCK), 
Macroprep-Methyl-HIC-Support.RTM., Macroprep t-Butyl-HIC-Support.RTM. (all 
from BioRad); 
TSK-Gel Butyl Toyopearl.RTM., TSK-Gel Phenyl Toyopearl.RTM. and 
TSK-Gel Ether Toyopearl.RTM. (all from Tosohaas) are used. 
A possible embodiment of the method according to the invention is 
illustrated in FIG. 3. 
The prothrombin preparation which can be prepared by the method according 
to the invention is not only characterised by an extremely high purity 
which is close to the theoretically possible purity of 10 U/mg, but also 
by the fact that even at a dose of at least 150 U of prothrombin/kg it is 
free from thromboembolic side effects, expressed by a score in the Wessler 
thrombisis model of 3 at the most, preferably 2 at the most, in particular 
less than 2, and therebeyond that as a lyophilisate it can be 
reconstituted with a reconstitution time of 1 min at the most to a clear 
solution having an activity of at least 10 U of prothrombin/ml up to 200 U 
of prothrombin/ml. The biological activity of the prothrombin preparation 
is to be understood as the enzymatic activity obtained upon activation of 
the prothrombin. 
According to a further aspect, the present invention relates to the use of 
purified prothrombinase factors, in particular of purified prothrombin 
and, optionally, purified factor Xa, for producing a pharmaceutical 
preparation for establishing supranormal prothrombin concentrations in a 
patient's blood, or to establish normal prothrombin concentrations in 
blood in case of conditions involving a lowered prothrombin level. 
It has been shown that with the preparations according to the invention 
such supranormal factor II levels can be maintained even permanently, i.e. 
over extended periods of time, which, on the one hand, goes back to the 
fact that for blood coagulation factors, prothrombin has a very high 
half-life as a medicament, and, on the other hand, also resides in the 
fact that the preparations according to the invention are free from side 
effects so that even a subcutaneous administration, e.g. by depot 
administration, is possible. The supranormal concentrations of prothrombin 
which are possible in blood by the administration of purified prothrombin 
and optionally purified factor Xa or other prothrombinase factors, amount 
to at least 150%, preferably even at least 200%, corresponding to an 
activity of at least 1.5 U of prothrombin/ml blood, preferably at least 
2.0 to up to 10 U/ml. 
Finally, the invention relates also to the use of purified prothrombinase 
factors, in particular of purified prothrombin and, optionally, purified 
factor Xa, for preparing a pharmaceutical preparation for the treatment of 
factor VIII inhibitor conditions, hemophilia A or B, and of von Willebrand 
Disease. It has been shown that in the animal model, with the preparations 
according to the invention a rapid and efficient treatment which is free 
from side effects is possible for all these indications. 
Preferably, the preparations according to the invention are provided in a 
solution having a physiological pH, which preferably does not contain any 
free calcium ions. However, it is also possible to use an acid buffer in 
the pH range of from 4.5-6.5, preferably 5-6, which is removed from the 
factor Xa activity optimum, by which the activation of prothrombin is 
avoided and therefore the stability of the combination preparation may 
once more be increased. It goes without saying that all the pharmaceutical 
additives and solutions suitable for factors II, V, Va, X and Xa may be 
used for the ready-to-use production of the preparations according to the 
invention. 
Furthermore, it has surprisingly been shown that with the preparations 
according to the invention even in non-hemophiliacs, acute bleedings, an 
increased bleeding intensity or an increased risk of bleeding may 
effectively be treated. In addition to the above-mentioned indications of 
the various types of hemophilia, i.e. hemophilia A and B as well as 
inhibitor hemophilia, the preparations according to the invention are also 
applicable in non-hemophiliacs. Among the non-hemophiliacs there are also 
those who have blood coagulation disorders based on inhibitors against 
blood factors which are not factor VIII or factor IX. Furthermore, 
patients can be treated who exhibit an impaired thrombin generation, which 
is caused by the absence or functional defect of one or several factors of 
the extrinsic or intrinsic coagulation or at the formation of antibodies 
to one or several of these factors or by a lack of the cellular receptor 
for one or several of these factors. Upon administration of a preparation 
according to the invention, a coagulation-promoting effect optionally may 
occur in vivo, whereby a treatment, i.e. a prophylactic or therapeutic 
administration becomes feasible. 
According to a further aspect, the present invention thus relates the the 
use of at least 2 coagulation factors which are components of a 
prothrombinase or of a pro-prothrombinase, or to the use of the 
preparation according to the invention for preparing a pharmaceutical 
preparation for the treatment of acute bleedings, an increased bleeding 
insensity or an increased risk of bleeding in non-hemophiliacs. 
In particular, conditions can be treated which are caused by an impaired 
aggregation behavior of blood platelets or thrombopathies, e.g. 
storage-pool defects, or due to a deficient or dysfunction of 
platelet-associated proteins, but also bleeding conditions caused by 
platelets deficiency (thrombocytopenia). One side effect of an 
anticoagulant therapy resides in the heparin-induced thrombocytopenia, 
which also constitutes an indication for the preparation according to the 
invention. 
According to a preferred embodiment, the present invention thus relates to 
the use of at least 2 coagulation factors which are components of a 
prothrombinase or of a pro-prothrombinase, respectively, for preparing a 
pharmaceutical preparation for the treatment of bleedings caused by a 
thrombocytopenia, in particular for the treatment of a heparin-induced 
thormbocytopenia. 
In this connection it is advantageous that the pharmaceutical preparation 
prepared according to the method of the invention may have a primary 
hemostatic activity. The efficacy of the pharmaceutical preparation 
prepared may be further enhanced by the combination with proteins having 
primary hemostatic activity. Primarily suited is a von Willebrand factor 
protein or a fraction of the von Willebrand factor, respectively, with a 
defined collagen binding activity. 
A further indication for the treatment of patients suffering from blood 
coagulation disorders is the prevention or treatment of bleedings 
occurring in connection with von Willebrand Disease. This disease with or 
without a prevalence of coagulation factor inhibitors involves an 
increased bleeding propensity or a risk of bleeding and in many cases it 
leads to bleedings which are difficult to control. By aid of the 
preparation according to the invention it is possible to quickly stop such 
a bleeding. 
Thus, according to the invention, a set for the treatment of patients 
suffering from blood coagulation disorders is provided, which comprises 
the following components: 
a) a pharmaceutical preparation comprising at least 2 coagulation factors 
which are components of a prothrombinase or of a pro-prothrombinase and 
which are free from phospholipids, and 
b) a protein having primary hemostatic activity, in particular vWF. 
Bleedings which are difficult to control may in some instances occur as a 
side effect of a therapy with synthetic, semi-synthetic and biological 
coagulation inhibitors or anticoagulants or thrombocyte function 
inhibitors. These substances interfere directly or indirectly with the 
coagulation system and may disturb the natural coagulation or clotting 
process in an undesired way. Thus, there is a demand for antagonists for 
these substances. In the prior art, the use of a prothrombin complex or of 
a FEIBA.RTM. preparation is known for the treatment of bleedings which 
have been caused by an anticoagulant therapy. In this respect, cf. Irani 
M. S. et al., The American Journal of Cardiology, Vol. 75, Feb. 15, 1995, 
p. 422; Fareed J. et al., Haemostasis 1991, Vol. 21 (suppl. 1), pp. 64-72; 
and Fareed J. et al., Seminars in Thrombosis and Hemostasis 1991, Vol. 17, 
No. 2, p. 137. 
According to a further embodiment of the present invention, the 
above-stated coagulation factors are used for preparing a pharmaceutical 
preparation according to the invention for the treatment of bleedings 
within the scope of an anticoagulant therapy, in particular for preparing 
a pharmaceutical preparation as an antidote for a coagulation inhibitor or 
for an anticoagulant or for a thrombocyte function inhibitor. What is 
substantial is that a risk of thrombosis involved with the presence of 
phospholipids is avoided. Particularly with patients who are subjected to 
a therapy with anticoagulants, there is an increased risk of thrombosis. 
According to the invention, however, the use of phospholipids can be 
obviated, whereby surprisingly the antidote effect becomes even more 
specific. 
The use according to the invention primarily relates to the antagonizing of 
a coagulation inhibitor or anticoagulant which indirectly or directly 
inhibit factor Xa or thrombin. According to the invention, preferably a 
substance is antagonized which is selected from the group consisting of 
APAP 
((2S)-2-4-(3S)-1-acetimidyl-3-pyrrolidinyl!-oxy!-phenyl!-3-(7-amidino-2 
-naphthyl)-propionic acid hydrochloride pentahydrate), benzamidine 
derivative, hirudin, heparin, heparin analogues, in particular 
pentasaccharides, AT III heparin complex, AT III, antistasin, 
"Tick-Anticoagulant Peptide", inactive coagulation factors, in particular 
"active site"-inhibited coagulation factors, TFPI, competitive ligands for 
thrombocyte membrane surface receptors, in particular antibodies to GP 
IIb/IIIa, and their genetically engineered or synthetically produced 
analogues, in particular peptides, as well as oral anticoagulants. Among 
the inactive coagulation factors there are primarily derivatised, mutated, 
fragmented, chemically or physically inactivated or inhibited factors of 
intrinsic and extrinsic blood coagulation, respectively, which may 
competitively interact with the native factor. 
The antagonizing of a thrombocyte-function inhibitor is primarily indicated 
in the case of ticlopidin or acetyl salicylic acid. 
Acute bleedings are primarily extremely critical in the region of the 
brain. Therefore, there is a need for the prevention or treatment, 
respectively, of intracranial bleedings, e.g. intraventricular hemorrhage 
(IVH). Particularly patients suffering from damaged blood vessels or from 
an impaired thrombin generation run an increased risk of intracranial 
bleedings. The preparation according to the invention is also specifically 
suitable for this indication, wherein above all the improved treatment of 
premature infants becomes possible. 
Within the scope of the use according to the invention at the indications 
listed, the respective kits are each provided which comprise the 
pharmaceutical preparation of the invention as one component. Furthermore, 
substances which intensify the hemostatic effect, e.g. a protein having 
primary hemostatic activity, in particular vWF, may be contained. Besides 
this, a kit for anticoagulant therapy naturally will contain the 
respective anticoagulant and the pharmaceutical preparation of the 
invention as an antidote.

EXAMPLE 1 
Preparation of factor X and factor II from a virus-inactivated plasma 
fraction by means of ion exchange chromatography. 
A lyophilized prothrombin complex factor preparation containing factors II, 
IX, X as well as protein C and protein S was prepared according to the 
method of Brummelhuis, H. G. J., Preparation of the Prothrombin complex, 
in Methods of Plasma Protein Fractionation, Curling, J. M. ed., 117-128, 
Academic Press, New York, (1980), and heat-treated for virus inactivation 
according to EP 159 311. Accordingly, the lyophilisate (1,000 U of factor 
X/g, 1,200 U of factor II/g) was dissolved in distilled water so that the 
latter contained 50,000 U of factor X/1, and adjusted to pH 7.0. After the 
addition of 12% (v/v) TWEEN.RTM. 80 it was stirred for 1 hour at room 
temperature. Subsequently, it was diluted 1:5 with a 20 mM 
Tris-HCl-buffer, pH 7.0, and the prothrombin complex protein fraction was 
adsorbed on calcium phosphate Ca.sub.3 (PO.sub.4).sub.2 ! at a 
concentration of 30 g Ca.sub.3 (PO.sub.4).sub.2 per 1 of prothrombin 
complex solution by stirring for one hour at room temperature. 
Subsequently, the solid phase was separated by centrifugation at 5,000 rpm 
for 20 min, and the precipitate was washed twice with 20 mM 
Tris-HCl-buffer, pH 7.0, containing 10% ammonium sulphate, by resuspension 
and renewed centrifugation. A third washing was performed in an analogous 
manner with 20 mM Tris-HCl-buffer, pH 7.0, containing 150 mM NaCl. Elution 
of the prothrombin complex fraction was performed with 1M sodium phosphate 
solution, pH 7.0, wherein 25 ml of this solution per g of calcium 
phosphate were stirred for 1 hour at room temperature and subsequently the 
remaining precipitate was separated by centrifugation as above. 
Subsequently, the supernatant was subjected to an ammonium sulphate 
precipitation with 366 g of ammonium sulphate per 1 for 15 h at 4.degree. 
C. under stirring. The precipitate which contained the prothrombin complex 
fraction was centrifuged off as above. The precipitate was taken up in a 
25 mM trisodium citrate dihydrate buffer, containing 100 mM NaCl, 1 mM 
benzamidine hydrochloride, pH 6.0, and re-buffered on a column filled with 
Sephadex.RTM. G-25 at 4.degree. C. with a linear flow of 1 cm/min against 
25 mM trisodium citrate dihydrate buffer, containing 100 mM NaCl and 1 mM 
benzamidine hydrochloride, pH 6.0, to separate the ammonium sulfate. 
Therein, UV absorption at 280 nm and electric conductivity were measured 
in the eluate flow. The protein-containing fractions were combined and 
subsequently subjected to an ion exchange chromatography over 
DEAE-Sepharose FF.RTM. (Pharmacia). The fractions were applied to a column 
(inner diameter: gel bed height=1:1.3) having a gel volume of 8.2 1, 0.55 
g of protein/l gel, at a linear flow of 0.36 cm/min. Chromatography was 
performed at 22.degree. C. Prior to application of the proteins, the 
column had been equilibrated with a 25 mM trisodium citrate dihydrate 
buffer, containing 100 mM NaCl, 1 mM benzamidine hydrochloride, pH 6.0. 
Elution of the protein fraction was effected in several stages with a 
buffer 1 (25 mM trisodium citrate dihydrate, 1 mM benzamidine 
hydrochloride, 245 mM NaCl, pH 6.0), a buffer 2 (25 mM trisodium citrate 
dihydrate, 1 mM benzamidine hydrochloride, 270 mM NaCl, pH 6.0), and a 
buffer 3 (25 mM trisodium citrate dihydrate, 1 mM benzamidine 
hydrochloride, 400 mM NaCl, pH 6.0). Elution with buffer 1 was carried out 
with 2.4 column volumes, inert protein being separated therewith. Elution 
with buffer 2 was carried out with 5.6 column volumes, and there fractions 
were collected which were analysed for their contents of factor II, factor 
X, protein C and factor IX. The factor X-containing fractions which were 
free from factor II, IX and protein C, were combined. By elution with 
buffer 3 (1.9 column volumes), factor II was desorbed, wherein again 
fractions were collected and assayed for their contents of factor X, 
factor IX and factor II. The factor II-containing fractions were pooled. 
Both, factor II and the factor X-containing pool optionally could be 
subjected to an additional treatment for inactivation of pathogenic 
impurities by the addition of 1M KSCN and incubation at 22.degree. C. for 
several hours. 
EXAMPLE 2 
Purification of factor II by means of hydrophobic interaction 
chromatography 
The factor II pool recovered in Example 1 was adjusted to 1.8M NaCl by the 
addition of sodium chloride, and the pH was corrected to pH 7.0. 
Subsequently, this solution was adsorbed to a gel, Phenylsepharose High 
Performance.RTM. (Pharmacia), by hydrophobic interaction, wherein 3 g of 
protein/l gel were bound. In a column having a ratio of inner diameter:gel 
bed height=1:1.9, the protein fraction was adsorbed at a linear flow of 
0.25 cm/min, and subsequently freed from inert protein by washing with a 
buffer (25 mM Tris-HCl, 3M NaCl, pH 7.4). Factor II was eluted from the 
column by gradient elution with 11.5 column volumes of 3M-0.9M NaCl with 
simultaneous collection of fractions, and those fractions which contained 
factor II activity but were free from factor X and factor IX, were pooled. 
Subsequently, the collected factor II fractions were concentrated ten-fold 
by ultra/diafiltration via an ultrafiltration membran having a cut-off of 
30 kD, and re-buffered against a buffer containing 4 g of trisodium 
citrate dihydrate/l, 8 g NaCl/l, pH 7.0. A thus prepared factor II 
preparation had a specific activity of 6.9 U/mg protein. The determination 
of the factor II activity was effected with the 1-stage method based on 
the thromboplastin time, by using a factor II-deficient plasma against the 
international factor II standard by using the reagent combination from 
IMMUNO, Vienna. In clotting analyses, other coagulation factors were 
detectable in traces or not at all (factor VII&lt;0.00002 U/U factor II, 
factor IX 0.0002 U/U factor II, factor X 0.004 U/U factor II, protein 
C0.003 U/U factor II, and factor VIII&lt;0.0002 U/U factor II). 
EXAMPLE 3 
Purification of factor II by means of hydrophobic interaction 
chromatography and hydroxyl apatite chromatography 
As an alternative preparation method for a highly purified factor II also a 
method was used, in which at first factor IX was separated from a 
lyophilized prothrombin complex factor preparation (cf. Example 1) by 
hydrophobic chromatography, subsequently factor II was isolated, and the 
latter was then highly purified by chromatography on hydroxyl apatite. The 
prothrombin complex factor preparation was dissolved as in Example 1 and 
incubated with a detergent at room temperature for 1 h. Subsequently, a 
factor II, IX and X-containing fraction was isolated as in Example 1 by 
ion exchange chromatography on DEAE-Sepharose FF.RTM. (Pharmacia). From 
this, the factor IX-containing fraction was subsequently removed by 
interaction with Butyl-Toyopearl.RTM. (Toso Haas). Subsequently, the 
adsorption supernatant was purified by a further hydrophobic interaction 
chromatography on Phenyl-Sepharose High Performances.RTM. (Pharmacia), 
wherein approximately 4 g of protein/l gel could be adsorbed. In a column 
having a ratio of inner diameter:gel bed height=1:1.9, the protein 
fraction was adsorbed at a linear flow of 0.25 cm/min, subsequently the 
inert protein was removed by washing with 20 mM Tris-HCl, 3M NaCl, pH 7.4, 
and finally the factor II-containing fraction was isolated by step-wise 
elution which desorbed from gel with decreasing conductivity at 1.9M NaCl. 
The factor II-containing fraction was then directly adsorbed on 
Ceramik-Hydroxylapatites.RTM. (Biorad). This was carried out on a column 
having a ratio of inner diameter:gel bed height=1:4.8. Elution was done at 
a linear flow of 3 cm/min. By elution with a salt gradient, factor II 
could be desorbed from the column. The factor II-containing fractions were 
collected and concentrated via ultra/diafiltration over polysulfon 
membranes with a cut-off of 30 kD, until the factor II concentration was 
50-100 U/ml. A thus prepared factor II preparation had a specific activity 
of at least 7 U/mg protein. Other coagulation factors, in particular 
factor IX and factor VIII, were detectable only in traces or not at all, 
as in the preparation from Example 2. By selection of a suitable 
diafiltration buffer, the factor II preparation was transferred into a 
pharmaceutically compatible buffer (e.g. 4 g of trisodium citrate 
dihydrate/l, 8 g NaCl/l, pH 7.0). 
EXAMPLE 4 
Recovery of factor Xa 
The factor X fraction prepared as described in Example 1 subsequently was 
further processed to factor Xab as described in DE 43 25 872, wherein the 
thus-obtained highly purified factor Xa preparation was lyophilized in the 
presence of 1 g/100 ml human albumin. Such a preparation was free from 
other clotting factors; the contained factor Xa had a specific activity of 
120 U/mg protein before the addition to albumin. 
EXAMPLE 5 
Lyophilization of factor II 
The preparation containing highly purified factor II described in Example 3 
was lyophilized without the addition of stabilizers, more than 80% of the 
initial activity remaining after lyophilization. 
EXAMPLE 6 
Colyophilization of factor II and factor Xa 
A factor II preparation prepared according to Example 3 was filled at a 
concentration of 100 U/ml at 20 ml into 50 ml bottles and shock-frozen at 
-80.degree. C. Subsequently, a solution of a highly purified factor Xa 
which had been prepared according to DE 43 25 872 and had a concentration 
of 500 U/ml was dosed in an amount of 30 .mu.l onto the frozen factor II 
solution. By immediate freezing of the small volume, a mixing of the 
factor II and factor Xa phases was prevented. Subsequently, it was 
lyophilized. To prepare the infusion solution, the lyophilzate was 
reconstituted with 20 ml A. dest., mixed, and immediately prepared for 
administration. 
EXAMPLE 7 
Pharmaceutical formulation of factor II, factor Xa and antithrombin III 
and/or antithrombin III heparin complex 
The highly purified factor II in combination with factor Xa and 
antithrombin III or antithrombin III heparin complex were diluted to the 
application concentration in a buffer containing 4 g of trisodium citrate 
dihydrate/l and 8 g NaCl/l, pH 7.0. These solutions could be lyophilized, 
an activity of at least 80% of the respective components remaining. 
EXAMPLE 8 
Detection of partial prothrombinase 
Formation of a complex of factor II and factor Xa to "partial 
prothrombinase" was demonstrated by the following experiment: 57 U of 
factor II from Example 3 and 1.2 U of factor Xa from Example 4 were 
dissolved in 20 mM Tris-HCl buffer, containing 150 mM NaCl, pH 7.4, and 
incubated for 15 min at room temperature for the formation of the complex. 
Subsequently, one aliquot of the solution was chromatographed by gel 
permeation chromatography over Superose.RTM. 12 (HR10/30) (Pharmacia) at a 
flow rate of 0.25 ml/min. The application volume was 200 .mu.l. The 
throughput through the column was measured UV-spectrophotometrically at 
280 nm and collected in fractions of 0.5 ml. Subsequently, factor Xa which 
was determined with a chromogenic substrate in a photometric test by means 
of the method described in DE 43 25 872 was measured quantitatively in the 
fractions. Likewise, the factor II activity was determined in the 
individual fractions as in Example 2. The result can be taken from FIG. 4. 
Factor Xa FIG. 4: - -.box-solid.- - Act. Xa (FII/Xa)! and factor II FIG. 
4: - -.circle-solid.- - Act. II.times.10(FII/Xa)! eluted together with the 
protein fraction FIG. 4:--A280 nm (FII/Xa)!. Under identical conditions, 
factor Xa then was applied alone to the column, the elution profile was 
determined after gel passage by measureing the UV absorption at 280 nm 
FIG. 4: .......A280 nM (FXa)! and factor Xa activity FIG. 4: - 
-.tangle-solidup.- - Act.Xa.times.10 (FXa)! was determined in the 
fractions. The protein peak corresponding to factor Xa was clearly offset 
from factor Xa in the complex with factor II. By reducing the retention 
time of factor Xa in the complex on the column and together therewith for 
displacement to an apparently higher molecular mass, it was possible to 
prove a complex formation of factor II and factor Xa (partial 
prothrombinase). 
EXAMPLE 9 
Formulation of the preparation in the double chamber syringe system 
To simplify the application of the multiple-component system, such as, 
e.g., mixtures of factor II and factor Xa or factor II, factor Xa and 
antithrombin III, a double double-chamber syringe body, as described in AT 
382 783, can be used as application device. In the clinical application of 
lyophilized multiple-component systems, the latter would otherwise each 
have to be reconstituted and mixed with each other at a defined ratio 
before being administered to the patient. Filling of the corresponding 
lyophilisate into a double-chamber syringe system allows for an exact 
dosing to a pre-determinable activity of the preparation for the treatment 
of inhibitor hemophilia, e.g. to conventional FEIBA units, which can be 
determined according to AT 350 726. By using such a system, the effective 
mixture is prepared in situ at the injection. In one embodiment, 
lyophilisates of the active substances, factor II and factor Xa, are each 
present in the two double-chamber syringe bodies, which, on account of the 
ready solubility of the highly purified proteins, solubilize by the 
addition of solvent and are immediately infused into the patient by 
further pressing down the syringe piston after mixing in the mixing head 
(cf. FIG. 1). 
On account of the high stability of factor II in solution, the former also 
is suitable as a solvent for factor Xa in the application form of a 
double-chamber syringe. A solution of highly purified factor II, prepared 
e.g. according to Example 2, in a physiologically compatible citrate 
buffer (4 g of trisodium citrate dihydrate/l, 8 g NaCl/l, pH 7.0) at a 
concentration of 100 U factor II/ml, is admixed with antithrombin III 
(IMMUNO, Vienna) (1 mU antithrombin III/U factor II). This solution is 
used as a solvent for the lyophilized powder of a highly purified factor 
Xa in a double-chamber syringe (cf. FIG. 2). 
EXAMPLE 10 
Stability of the highly purified factor II 
Factor II was purified as described in Example 2 and stored as a solution 
at a concentration of 60 U/ml in a buffer containing 4 g/l trisodium 
citrate dihydrate, 8 g/l NaCl, pH 7.0, at 5.degree. C., at 22.degree. C., 
at 37.degree. C. and at 50.degree. C. At the storage temperatures of 
5.degree. C. and 22.degree. C., samples were drawn every 24 h, at the 
storage temperatures of 37.degree. C., and 50.degree. C., samples were 
taken over 24 h at 1 h, 2 h, 4 h, 8 h and 24 h. In each sample the factor 
II activity was determined. 
At 5.degree. C. more than 80% of the initial activity could be detected 
even 86 days after storage had been started. At 22.degree. C., more than 
80% of the initial activity was found for 14 days; at 37.degree. C., 95% 
of the initial activity could be found 24 h after storage had been 
started. Even at 50.degree. C., 94% of the original activity could still 
be found 8 h after storage had been started. 
EXAMPLE 11 
Autoactivation and stability of the preparations according to the invention 
Inventive preparations according to Example 5 (factor II) and Example 6 
(factor II/Xa complex) were examined in an in vitro-test for their 
prothrombotic properties particularly of the extrinsic clotting system, as 
compared to two commercially available prothrombin complex concentrates. 
Before being used in the test, the heparin present in the prothrombin 
complex concentrate was neutralized with protamin sulphate corresponding 
to the concentration (of heparin) so as to exhibit thrombogenic substances 
that would be masked by heparin. 
The analysis material consisted of 250 .mu.l Thrombotest.RTM. (Nycomed 
Pharma AS, Oslo, Norway), a preparation containing thromboplastin from 
bovine brain and adsorbed bovine plasma, which was pre-incubated for 3 min 
at 37.degree. C. Subsequently, 50 .mu.l of a sample were added, and the 
clotting time was determined by means of a spherical coagulometer (KC4, 
Amelung). Bovine thromboplastines are considered to be particularly 
sentitive to activated clotting factors. Accordingly, the test system can 
provide information on the in vitro-thrombogenicity of the preparations 
assayed. In this test, undiluted normal human plasma was used as a 
control. The latter had a clotting time of 74 seconds. When used 
undiluted, the two assayed preparations of the invention had clotting 
times of more than 100 seconds, the concentration corresponding to a 
possible application concentration of 30 U of factor II/ml. A commercially 
available prothrombin complex concentrate, likewise dissolved in the 
application concentration and diluted to 30 U/ml, had to be further 
diluted with buffer 1:32 to achieve the clotting time of normal plasma (74 
seconds). A further commercially available, activated prothrombin complex 
concentrate even had to be diluted with buffer 1:216 so as to achieve the 
clotting time of normal plasma, i.e. 74 seconds. Subsequently, the 
preparations according to the invention were stored in the dissolved state 
at room temperature for up to 4 h and samples were taken once per hour, 
and again the clotting time was determined by means of Thrombotest.RTM.. 
No change had taken place, i.e. even after 4 h the samples used in the 
undiluted state still have clotting times of more than 100 seconds. From 
these tests it could be seen that the preparations according to the 
invention have a markedly lower thrombogenicity potential as compared to 
commercially available prothrombin complex concentrates. 
EXAMPLE 12 
Dissolution behavior of the preparation according to the invention 
A highly purified factor II-containing preparation was prepared as 
described in Example 5 and lyophilized. The lyophilisate was adjusted such 
that the volume activity was 50 U/ml after reconstitution with distilled 
water. This was a typical application concentration. On account of the 
high purity of the preparation, however, also volume concentrations of 100 
and more units of factor II per ml could be achieved. The dissolution time 
of the preparation, defined as that time which passes from the addition of 
the solvent (distilled water) until the complete dissolution of the powder 
was determined. In comparison thereto, the lyophilisates of two 
commercially available preparations, i.e. prothrombin complex and 
activated prothrombin complex, were each reconstituted according to the 
supplier's instruction at the application concentration by the addition of 
distilled water, and the dissolution times were also determined. The 
dissolution times of the tested preparations can be taken from the 
following table: 
______________________________________ 
Preparation of 
the invention 
APCC.sup.1 
PCC.sup.2 
______________________________________ 
Dissolution time (s) 
&lt;30 240 300 
______________________________________ 
.sup.1 APCC = activated prothrombin complex concentrate 
.sup.2 PCC = prothrombin complex concentrate 
When the respective preparations had been dissolved, an UV/VIS spectrum 
between 280 and 750 nm was measured of each of them, against a citrated 
saline buffer. The spectra can be taken from FIGS. 5a, b and c (a: 
preparation according to the invention; b: activated prothrombin complex; 
c: prothrombin complex). A comparison of the spectra showed that the 
preparation according to the invention had a markedly lower light 
absorption in the visible range up to 700 nm than the preparations of 
comparison. The preparation according to the invention thus was marked by 
an excellent solubility and, in the dissolved state, was present as a 
clear, uncolored solution, as is characteristic of an improved 
pharmaceutical preparation. 
EXAMPLE 13 
Effect of the preparation in factor VIII inhibitor plasma 
A high-titer factor VIII inhibitor plasma (55 BU/ml) was pre-cooled in an 
ice bath and incubated for one minute and at 37.degree. C. with PTT 
reagent (IMMUNO, Vienna) and the sample to be tested, both also pre-cooled 
in an ice bath, at a ratio of 1+1+1. Subsequently, 1 part of an 0.05M 
CaCl.sub.2 solution was added, and the clotting time of the test mixture 
was determined by means of a spherical coagulometer (Amelung), model KC-4. 
The preparations to be tested further diluted 1:10 in a buffer comprising 
7 g NaCl/l and 6 g of trisodium citrate dihydrate/l, and used for the test 
at this concentration. Under these conditions, highly purified factor II, 
alone and in combination with highly purified factor Xa, in combination 
with antithrombin III and heparin was tested. The clotting times can be 
taken from the following table. 
______________________________________ 
Test substance 
(concentration) Clotting 
Factor II 
Factor Xa 
Antithrombin III 
Heparin 
time (sec) 
______________________________________ 
10 U/ml -- -- -- 147 
10 U/ml 0.1 U/ml -- -- 63 
10 U/ml 0.1 U/ml 1 U/ml -- 55 
10 U/ml 0.1 U/ml 1 U/ml 6 U/ml 
49 
______________________________________ 
As the control, the clotting time of the test sample was determined with 
pure sodium chloride trisodium citrate buffer. The latter was 148 seconds. 
Thus, the clotting time of the inhibitor plasma could be markedly reduced 
merely by the addition of factor II/Xa with or without antithrombin III or 
heparin. By measuring a FEIBA standard preparation (FEIBA STIM 4, IMMUNO), 
which is the conventional preparation for the treatment of factor VIII 
inhibitor patients, at various concentrations in the same test sample it 
could be found that the FEIB-activity of the preparation containing 10 
U/ml factor II and 0.1 U factor Xa/ml corresponds to approximately 25 U 
FEIBA/ml. 
EXAMPLE 14 
In vivo-effect with factor VIII inhibitor hemophilia 
To test the in vivo efficacy of the preparation according to the invention, 
a factor VIII inhibitor hemophilia rabbit model was used. White New 
Zealand rabbits weighing approximately 2 kg each were anesthesized. After 
the onset of anesthesia, each right femoral vein was prepared and a 
permanent venous access was established. Through the latter, per kg body 
weight, 0.5 ml of a human factor VIII inhibitor plasma (1500 BU/ml) were 
infused during 10 min. 30 min after the end of infusion, the bleeding 
characteristic was determined by using a modified method according to 
Giles et al., Blood 60: 727-730 (1982). For this, the fur surrounding the 
claw of a hind paw of the rabbit was shaved to prevent blood emerging at 
the later bleeding from being absorbed by the fur. The nail cuticle was 
injured by means of a nail clipper; immediately thereafter, filters were 
placed therebelow such that the blood could drip directly onto the filter 
without being sucked up thereby on account of a capillary effect, so as to 
prevent a forming blood clot from being destroyed. The filter units were 
exchanged every 2 minutes, and the emerging blood was collected in 
fractions. Blood was continued to be collected for 30 minutes, and then 
the wound was cauterized if the bleeding had not stopped. To quantitate 
the bleeding characteristic, the filters were each extracted with 0.04% 
ammonium hydroxide solution for 5 h, whereby the erythrocites, which had 
been collected in the filter with the blood, were lysed. By a 10 min 
sonication, the hemoglobin was extracted and quantitated photometrically 
at 416 nm against a calibration curve. The latter was established by 
pipetting rabbit blood in volumes of between 10 .mu.l and 1 ml onto 
filters, extracting the filters as described above and photometrically 
determining the hemoglobin at 416 nm. The bleeding characteristics of the 
nail cutting was determined by graphically plotting the amounts of blood 
per 2 min fraction against time. For an evaluation, the accumulated blood 
loss was determined by graphically plotting the volume of the individual 
blood fractions against time. The slope of the cumulative bleeding between 
10 and 20 min was taken as a relevant bleeding criterion. This value was 
independent of the initial blood amount which was subject to variations 
from rabbit to rabbit, depending on the claw cut. The increase of the 
bleeding characteristic in 10 to 20 min observation intervals served as a 
measure for the intensity of bleeding. An increase equal zero meant that 
the bleeding had stopped, an increase &gt;0 with a correlation coefficient of 
&gt;0.8 meant that there was a constant bleeding. Under the test conditions, 
healthy rabbits had a bleeding intensity of &lt;2 .mu.l blood/min. Factor 
VIII inhibitor rabbits exhibited a bleeding intensity of approximately 50 
.mu.l blood/min (cf. FIG. 6). Factor VIII inhibitor rabbits which had been 
treated with a factor II preparation according to Example 3 at a dosage of 
75 U/kg body weight as an infusion over 30 min showed a mean (n=6) 
bleeding intensity of 9.3 .mu.l/min and thus a significant reduction as 
compared to the untreated inhibitor animal. 
EXAMPLE 15 
In vivo effect in case of von Willebrand factor/factor VIII inhibitor 
Analogous to Example 14, a von Willebrand factor/factor VIII inhibitor 
model was established by infusing rabbits with an anti-von Willebrand 
factor/factor VIII antiplasma from goat which had been recovered by 
immunization of goats with a purified factor VIII/von Willebrand factor 
preparation, at a dosage of 1 ml/kg body weight. Such pre-treated animals 
exhibited an increased intensity of bleeding (cf. FIG. 7). The bleeding 
characteristics were measured by claw cuts performed on these animals, 
simultaneously with an infusion of the test substance and 30 min after 
infusion of the test substance had ended. By administering factor II as a 
bolus of 2.5 ml at a dosage of 75 U/kg, the initially increased bleeding 
intensity could be reduced from 77 .mu.l/min to 31 .mu.l/min (1st claw 
cutting) and 12 .mu.l/min (2nd claw cutting), respectively. By 
combining-factor II (75 U/kg) with a factor Xab (prepared according to 
Example 4) at a dosage of 0.55 U/kg as a bolus, at an injection volume of 
2.5 ml, the propensity to bleeding could be reduced from the initial value 
to 18 .mu.l/min at the first claw cutting and 5 .mu.l/min at the 2nd claw 
cutting. Thus, the abnormally increased bleeding behavior of the inhibitor 
animals was normalized to the level of the healthy control animals (4 
.mu.l/min). 
In addition, also the combination of factor II (75 U/kg), factor Xa (0.55 
U/kg) and antithrombin III (IMMUNO Vienna) (75 mU/kg) was examined in the 
same model. At the first claw cut, a reduction to 35 .mu.l/min, and at the 
second claw cut, to 7 .mu.l/min could be achieved (cf. FIG. 8). The 
addition of antithrombin III should prevent a generation of thrombin from 
prothrombin due to the enzyme factor Xa in the administered solution. 
EXAMPLE 16 
Thrombogenicity of the preparation according to the invention 
The individual components and mixtures thereof, of the preparation 
according to the invention were tested for their thrombogenic activity by 
using the method described by Wessler, J. Appl. Phys. 14: 943-946 (1959) 
in the venous blood subject to stasis in rabbits. 
Rabbits were anesthesized with pentobarbital, the vena jugularis of the 
animals was dissected and provided with loose ligatures at a distance of 
1-2 cm. The substances to be tested were injected into the animals, into 
the ear vein opposite the dissected vena jugularis. The injection was 
effected within 15 seconds. After a waiting time of 10-15 seconds, the 
vein segment was clamped. After further 10 minutes, the clamped vein 
segment was removed and cut open in a citrated buffer in a Petri dish, and 
the thrombi obtained were evaluated between 0 and 4 by means of a score 
(cf. table) 
______________________________________ 
Degree of Thrombosis Score 
______________________________________ 
no formation of thrombi 
0 
few small thrombi 0.5-1 
few middle-sized and many small thrombi 
2 
many middle-sized thrombi 
3 
few large thrombi 3.5 
one coherent thrombus 4 
______________________________________ 
Each substance was tested on six animals each receiving 75 U of factor 
II/kg, 0.55 U of factor Xa/kg and 75 mU of antithrombin III/kg either 
separately or in combination. The following table shows the mean value of 
the Wessler score of six examined animals each. Pure citrated buffer which 
was also used as a dilution buffer was used as the control. 
______________________________________ 
Test Substance 
Factor II 
Factor Xa Antithrombin III 
Wessler Score 
______________________________________ 
+ - - 0.17 
- + - 0.17 
- - + 0.08 
+ + - 0.25 
+ - + 0.08 
- + + 0.17 
+ + + 0.17 
- - - 0.2 
(Buffer) (Buffer) (Buffer) 
______________________________________ 
It proved that none of the components used, neither separately nor in 
combination, had a thrombogenic activity in rabbit. 
EXAMPLE 17 
Action of the preparation according to the invention in dependence on the 
form of administration 
A preparation according to Example 6 which contained factor II and factor 
Xa was each tested for its efficacy in 6 rabbits with induced factor VIII 
inhibitor hemophilia (cf. Example 14). The dose examined was as described 
in Example 15. As the control, the animals were infused with pure buffer. 
In the first test, an infusion of the test substances was carried out for 
30 minutes, corresponding to approximately 15 min/kg body weight, by means 
of an automatic infusion pump, at an infusion rate of 1 ml/min. In a 
second test, the same dose, yet given as a bolus within 30 seconds in a 
small injection volume of 2.5 ml/kg body weight was administered to the 
animals. As in Example 14, the bleeding intensity was measured during and 
after substance administration. It proved that both, at slow infusion with 
a large infusion volume, and also at rapid injection of a small volume 
dose, but with identical doses relative to the body weight, the 
pathologically extended bleeding intensity of 67 .mu.l/min could be 
normalized to 5 .mu.l/min, which corresponds to the bleeding behavior of 
the healthy control animals. By administration of buffer no change of the 
bleeding intensity was found with either of the two infusion modalities. 
Despite the rapid injection as a bolus, no intolerance reaction could be 
observed during injection, and neither so during slow infusion. For 
commercially available preparations of comparable indication (activated 
prothrombin complex preparations), infusion rates in the range of &lt;0.05 
ml/min/kg body weight are recommended to avoid acute thromboembolic side 
effects and intolerance reactions. As the test indicated, due to its high 
purity the preparation of the invention could be administered without any 
side effects at 5 ml/min/kg body weight, i.e. at the 100-fold injection 
rate. 
EXAMPLE 18 
Action of partial prothrombinase in rats exhibiting a platelet defect 
Fawn-Hooded rats described by T. B. Tschopp and M. B. Zucker (Hereditary 
Defect in Platelet Function in Rates, Blood 1972; 40: 217-226) are used as 
the thrombocytopathy model. This rat strain which had been further 
characterized by S. L. Raymond and W. J. Dodds (Characterization of the 
Fawn-Hooded Rats as a Model for Hemostatic Studies, Thrombos Diathes. 
haemorrh. 1975; 33: 361-369) is characterized by an abnormally increased 
bleeding time and a reduced platelet aggregation, while prothrombin time, 
partial thromboplastin time, plasma factor VIII and fibrinogen levels as 
well as platelet number are in the same range as that of the healthy rat. 
The thrombopathy occurring in the fawn-hooded rat is associated with a 
reduced thrombin-induced ATP and ADP release, as well as with a reduced 
serotonin release and thus is characterized as "storage pool deficiency" 
reflecting the "storage pool deficiency" in man. 
The fawn-hooded rats are anesthesized with ketamine- xylazine (100 mg/kg+5 
mg/kg) i.m.. A catheter is placed into a jugular vein, via which the 
substances to be tested are infused. Subsequently, the bleeding time is 
determined, Simplate R disposable blood lancet (Organon, Technica) being 
used therefor. At a distance of approximately 1.5 cm from the radix of the 
tail, one dorsal, and subsequently one ventral longitudinal section are 
made and the bleeding time is measured. The mean value of the two 
individual measurements is taken as the bleeding time. Then the test 
substance (buffer, factor II according to Example 2, factor Xa according 
to Example 4, and partial prothrombinase according to Example 6) is 
applied; after 30 minutes, the bleeding time is determined again. Healthy 
Sprague-Dawley rats (Charles River) are used as control. 
Testing was effected in groups of 10 animals each, the complex of the 
partial prothrombinase being administered in 2 doses, 75 U/kg body weight 
(BW) prothrombin and 0.55 U/kg BW factor Xa as well as 150 U/kg BW 
prothrombin and 1.1 U/kg BW factor Xa. As the control, the individual 
components and buffer are administered. The mean bleeding times measured 
in the individual test groups are given in the following table. Healthy 
rats have a bleeding time of 168.+-.5 seconds under the same test 
conditions, while rates suffering from thrombopathy have an increased 
bleeding time of 335.+-.10 seconds. By administering partial 
prothrombinase, this abnormally increased bleeding time can be reduced to 
approximately 270 seconds. 
TABLE 
______________________________________ 
Bleeding time (sec) 
Mean value from Factor II (U/kg) 
10 animals 0 75 150 
______________________________________ 
FXa 0 335 .+-. 10 
296 .+-. 13.sup.ns 
307 .+-. 11.sup.ns 
(U/kg) 0.55 n.d. 273 .+-. 13* 
n.d. 
1.1 320 .+-. 13.sup.ns 
n.d. 268 .+-. 11*** 
______________________________________ 
Statistical evluation: 
The data are given as mean values .+-. standard deviation. 
The group mean values were compared by means of the Student T test. 
n.d. not determined 
ns Difference to control group (0 U/kg FII and 0 U/kg FXa) not significan 
*Difference to control group (0 U/kg FII and 0 U/kg FXa) significant with 
p .gtoreq. 95% 
***Difference to control group (0 U/kg FII and 0 U/kg FXa) significant 
with p .gtoreq.99.9%. 
EXAMPLE 19 
Action of partial prothrombinase in von Willebrand factor-deficient dog 
A dog exhibiting congenital von Willebrand factor deficiency having 
non-measurable von Willebrand factor activity and antigen plasma levels 
and a factor VIII plasma concentration that is reduced by 50% is treated 
with a partial prothrombinase at a dose of 100 U/kg BW. For this, the dog 
is anesthesized and subsequently the partial prothrombinase, produced 
according to Example 6, is given intravenously as a bolus. Immediately 
before administration of the test substance as well as 15 min, 30 min, 1 
h, 2 h, 3 h, 24 h and 48 h after infusion, blood samples are taken, plasma 
is produced therefrom, and the prothombin, thrombin and factor VIII 
concentrations are determined in the plasma samples. 
Before administering the partial prothrombinase as well as 3 and 24 h after 
injection, the cuticle bleeding characteristics are determined. For this, 
the method of A. R. Giles, S. Tinlin and R. Greenwood, A Canine Model of 
Hemophilic (Factor VIII:C Deficiency) Bleeding, Blood 1982; 60, is used in 
a modified form. The fur surrounding the claw is shaved to prevent blood 
emerging at the later bleeding from being absorbed by the fur. The cuticle 
is injured by means of a nail clipper. Immediately thereafter, filters 
(Pipetman P5000 protection filter, Gilson) are established below the wound 
such that blood can drip directly onto the filter without being sucked up 
thereby on account of a capillary effect, so as to prevent a forming blood 
clot from being destroyed. The filter units are exchanged every 2 minutes, 
and the emerging blood is collected in fractions. Blood is continued to be 
collected for 30 minutes, and then the wound is cauterized if the bleeding 
has not stopped. Different claws of one and the same animal can be used. 
To quantitate the bleeding characteristic, the blood collected in fractions 
on the filters is each extracted with 5 ml of a 0.04% ammonium hydroxide 
solution for 5 h. Thereby the erythrocites, which have been collected in 
the filter with the blood, are lysed. By a 10 min sonication (Sonorex RK 
100, Bandelin electronic, Berlin), the hemoglobin is extracted and 
quantitated photometrically at 416 nm against a calibration curve. A 
calibration curve can be established by pipetting canine blood in volumes 
of between 10 .mu.l and 1 ml onto filters, extracting the filters as 
described above and photometrically determining the hemoglobin at 416 nm. 
Correspondingly, linear calibration curves can be established which enable 
the hemoglobin concentration to be calculated directly for the amount of 
blood per filter. The bleeding characteristics of the cuticle cutting are 
determined by graphically plotting the amounts of blood per 2 min fraction 
against time. For an evaluation of the bleeding characteristics, the 
accumulated blood loss is determined by entering in the graph the 
individual blood fractions additively against time. The increase of the 
accumulated bleeding between 10 and 20 min is taken as a relevant bleeding 
criterion. This value is independent of the initial blood amount which may 
be subject to variations because of poorly standardizable claw cutting 
techniques. The increase of the bleeding characteristic in 10 to 20 min 
observation intervals serves as a measure for the intensity of bleeding 
and is stated in ml blood/min. An increase equal zero, corresponding to 0 
ml/min, means that the bleeding has stopped; an increase &gt;0 with a 
correlation coefficient of &gt;0.8 means that there is a constant bleeding. 
Normal dogs tested under these conditions do not exhibit a bleeding in the 
observation interval, i.e. the bleeding has already previously come to a 
stop (bleeding intensity: 0.0 ml/min). 
Prior to administration of the partial prothrombinase, the bleeding 
intensity was 1.05 ml/min, and after 3 h it was reduced to 0.35 ml/min, 
and after 24 h it was further reduced to 0.47 ml/min. 
The factor VIII plasma level remained constant over the entire period of 
observation, von Willebrand factor antigen remained below the detection 
limit, prothrombin was at 0.7 U/ml before substance administration and 
rose to 2.4 U/ml after injection of the partial prothrombinase and was 
eliminated from circulation with a half-life of approximately 24 h. 1 h 
after administration of the test substance, a significant thrombin 
generation could be determined with a chromogenic substrate for thrombin 
Th1 (IMMUNO). Injection of partial prothrombinase was tolerated by the dog 
without any side effects. 
EXAMPLE 20 
Action of partial prothrombinase and of von Willebrand factor in the von 
Willebrand factor-deficient dog 
Analogous to Example 19, the dog was treated with a partial prothrombinase 
at a dosage of 100 U/kg BW and a purified von Willebrand factor at a 
dosage of 60 RCoF U/kg BW. Administration of this combination lead to a 
complete normalization of the abnormally increased bleeding behavior after 
24 h in the von Willebrand factor-deficient dog, the plasma parameters 
being comparable as in Example 19 relative to prothrombin and thrombin. 
Therebeyond, there was an endogenous factor VIII increase to approximately 
200% of the initial value, which remained constant over a period of more 
than 48 h and subsequently returned to the initial value over a period of 
approximately 120 h. Von Willebrand factor was eliminated with a half-life 
of 20 h. From the literature (L. Drouet, J. Roussi, M. Bonneau, G. A. 
Pignaud, P. L. Turecek, F. Dorner, U. Schlokat, F. G. Falkner, B. Fischer, 
A. Mitterer and H. P. Schwarz, The effect of recombinant human von 
Willebrand Factor in pigs with severe von Willebrand Disease. Blood 1995; 
86: 612a-AbsNo.2435 (abs)) it had been known that the administration of 
von Willebrand factor alone leads to an only partial normalization of the 
bleeding behavior. 
EXAMPLE 21 
Action of partial prothrombinase as an antagonist of peptide anticoagulants 
A partial prothrombinase in a composition of 57 U of factor II and 1.2 U of 
factor Xa was dissolved in 20 mM Tris-HCl-buffer containing 150 mM NaCl, 
pH 7.4, and incubated for 15 min at room temperature for the formation of 
the complex. Subsequently, an aliquot of 50 .mu.l was taken and incubated 
for 90 seconds at 37.degree. C. with 50 .mu.l of a Tris-imidazole buffer, 
pH 8.4. Subsequently, it was admixed with 100 .mu.l of a solution of the 
chromogenic substrate methoxy-carbonyl-D-cyclohexyl 
alanyl-glycyl-L-arginine-p-nitroanilide-hydroacetate so that the 
concentration of the chromogenic substrate was 1 mmol/l. Subsequently, the 
kinetics of the cleavage of the chromogenic substrate was photometrically 
determined at 405 nm for 3 min at 37.degree. C. The chromogenic substrate 
which is hydrolyzed by both thrombin and factor Xa, had a .DELTA.OD/min of 
0.084 when incubated with partial prothrombinase. As a control, factor Xa 
of the same concentration, yet without prothrombin was used in this test, 
and there appeared a conversion rate of the chromogenic substrate of 0.064 
.DELTA.OD/min. Recombinant hirudin (Rhein-Biotech) at a concentration of 
0.0025 U/ml was added to the partial prothrombinase, and the conversion 
rate of the chromogenic substrate was also investigated. It showed that no 
further substrate conversion could be measured after the deduction of the 
substrate conversion of pure factor Xa (0.064 .DELTA.OD/min), which was 
due to a complete neutralisation of the hirudin. 
Beside the effective and specific thrombin inhibitor hirudin, then also the 
selective factor Xa inhibitor, recombinant Tick-anticoagulant peptide, a 
recombinant equivalent of serin protease inhibitor of ornithodoros moubata 
whose anticoagulatory in vivo-efficacy had been described by G. P. Vlasuk, 
D. Ramjit, T. Fujita et al. in Comparison of the In Vivo Anticoagulant 
Properties of Standard Heparin and the Highly Selective Factor Xa 
Inhibitors Antistasin and Tick Anticoagulant Peptide (TAP) in a Rabbit 
Model of Venous Thrombosis. Thrombos Haemostas 1991; 65: 252-262, was 
added at a concentration of 50 .mu.g/ml. Already 30 min after the addition 
of the recombinant tick-anticoagulant peptide, no enzyme activity could be 
measured any longer with the chromogenic substrate. The test shows that 
partial prothrombinase is an efficient antagonist of the peptide 
anticoagulants hirudin and tick-anticoagulant peptide.