Process for producing a factor v concentrate

The invention relates to a method for preparing a factor V-concentrate from human blood plasma or a fraction of blood plasma containing an anti-coagulant.

To the biological phenomenon of hemostasis contribute the vessels, platelets and plasmatic factors. The primary wound closure caused by the synergistic interaction of vessels and platelets, leading to the so-called primary wound closure, which is then solidified by the plasmatic coagulation, leading to the formation of fibrin. Platelets form the reaction surface for the flow of plasma coagulation, at 13 plasmatic factors inhibitors not counting farther. These plasmatic factors are proteins, mainly glycoproteins in their effect to mainly serine proteases. These circulate as inactive precursors in the blood and to stimuli as they violate activated. The activation takes place by the principle of a limited proteolysis in which the foregoing in the coagulation cascade factor the respectively activated on it following the principle of an enzyme-substrate reaction. Essential for the activation are phospholipids such as are found in the form of a mixture, the so-called platelet factor 3 (PF 3) in the platelets and calcium ions. In addition, so-called accelerators important proteins that are able to accelerate the course of the coagulation. Among the accelerators, which act in conjunction with phospholipids and calcium, by forming so-called activation complexes include factors V and VIII (FV and F VIII).

FV is a β-globulin having a molecular weight of 330,000 Dalton, which activates a limited proteolysis by thrombin and by protein C_a is inactivated. This creates specific activation peptides.

The effect of FV as accelerator is due to the fact that the interaction between FX a and prothrombin in the presence of calcium and Plättchenphospholipiden intensified so that the thrombin generation is accelerated about 1000-fold. The complex of factors Xa, phospholipids, calcium, and FV a is called prothrombin activator or prothrombinase. According to the Akzeleratorwirkung of FV a reduction of this clotting factor leads to a slowing of thrombin formation and thus the coagulation and as a biological consequence to an increased bleeding risk.

All clotting factors, regardless of their enzymatic nature, are comparatively very labile and denaturierungsempfindlich, at least in the form in which they are usually obtained from anticoagulated plasma. In particular degree this is true for the F VIII and the FV, the two Akzeleratorproteine.

From the instability of factor V to do in the production of F V-deficient plasma use: citrated human plasma, is incubated in a water bath at 37 ° C, loses within 24 hours of its F V-type, all other factors remain stable ,

Congenital and acquired FV deficiency states are relatively rare. Nevertheless, there are a number of indications where an F V concentrate appears indexed. Due to the instability and Denaturierbarkeit is still, however, such a concentrate unavailable. This is quite in contrast to the other coagulation factors, all of which are in the form of concentrates for the therapy. This illustrates the particular instability of this clotting factor, which has been reflected in the label "unstable factor" for factor V.

For these reasons, there was a need for a process for the recovery of FV from whole blood, which guarantees the stability of this factor during the acceptance, storage and processing of the blood in the presence of anticoagulants. Such stabilization of FV would enable the production of a stable F-V plasma for clotting analytical studies as well as of F V-containing plasma for substitution or as a starting material for the production of an F V concentrate.

The object of the invention is the preparation of factor V-concentrate.

Although the FV is easy denaturierbar, he has already been isolated from plasma in active form. This was possible by synthetic protease inhibitors zusetzte fresh citrate plasma from which the prothrombin were removed as protection against enzymatic degradation of the FV. For the manufacture of a standard plasma and also for the preparation of pure FV such additives do not qualify because they are toxic and block the interaction of FV with other clotting factors.

It is also already known, factor V to win by chromatography on an anion exchanger of plasma from which the cryoprecipitate was separated. A disadvantage of this method is the low yield.

Kane WH et al, The Journal of Biological Chemistry, Vol 256, No. 2, 1981, pages 1002 -.. 1003 describes a method for the isolation of factor V from an anticoagulant-containing fraction from human blood plasma using calcium chloride. According to this method contained in the citrate plasma factor V is purified by adsorption to barium in the presence of 11% polyethylene glycol partially and then by DEAE-^RSepharose and ^RUltrogel AcA 34 chromatography isolated. Äquilibrier- or elution solvents are used for the chromatographic separation of Factor V containing 5 mmol / L calcium chloride, as this stabilizes the factor V.

Knows. HJ Thrombos. Diathes, Haemorrh, Volume 14, No. 32, 1965, pages 32 -. 51 indicates that the lability of factor V increases with the concentration of anticoagulants that calcium plasma cation seems to be that the Factor V from loss of activity in vitro protects and that various divalent cations may stabilize the factor V as a function of the type of anticoagulant used.

Blomback B., Nature, vol 198, Nr. 4883 shows the stabilizing effect of calcium (0.1 mM) on the activity of factor V in plasma fractions partially purified from citrated plasma.

It was surprisingly found that the loss of factor V activity at a plasma generation from a function of citrate concentration is, if citrate is used as anticoagulant. While FV is relatively stable at 20 ° C and 5 mmol / l citrate, it comes with increasing citrate concentrations in denaturation. A citrate concentration of 5 mmol / L could not be reached, when a coagulation should be avoided.

Surprisingly, it was further found that an activity loss can be reduced by the addition of divalent cations, preferably calcium ions. While the F V activity in citrated plasma, which usually contains tri-sodium citrate at a concentration of 11 mmol / l, at 37 ° C after 21 hours to values ​​falls nache 0% of normal, 35% of the activity is retained when such a solution is 2.5 mmol / l calcium ion, and 70% when it contains 5 mmol / l calcium ions.

The invention relates to a process for preparing a concentrate of coagulation factor FV from an anticoagulant-containing human blood plasma or blood, characterized in that one adds such a plasma or blood, a salt of calcium, magnesium or manganese in an amount such that a concentration of divalent cations of these metals is accomplished by at least two mmol / l but less than the concentration of the anticoagulant in a known manner equivalent concentration and wins an F V concentrate.

Preferably, such a metal salt is a soluble salt of calcium, especially calcium chloride or -azetat.

The concentration of divalent cations is at least 2 mmol / l. The effect does not increase steadily with the concentration, but can pass through a maximum. The concentration must not of course be so great that the mixture coagulates.

Anticoagulants are mainly salts of citric acid, heparin, phosphate or citrate dextrose, or mixtures of these.

The divalent cations must not be added immediately after removal of the blood. It is an advantageous effect is even still achieved when these are added up to 12 hours after blood collection, particularly when the plasma was stored at 3-5 ° C for so long. An addition of such cations to again frozen and thawed plasma or to the supernatant, after separation of the cryoprecipitate also significantly stabilized the F V.

Fig. 1 shows the dependence of F V activity of the time and the calcium concentration. The pH was as citrate 7.5-7.8, the temperature 4 ° C.

To this was human citrate frozen plasma, which was obtained by freezing a mixture of 9 parts by volume of whole blood and one volume part of sodium citrate and having a Citratendkonzentration of 10 mmol / l, thawed and against a physiological buffer having a citrate concentration of 10 mmol / l and calcium concentration of 0.25, 0.5, 1 or 2 mmol / l dialyzed.

In FIG. 1 corresponds to a concentration of calcium of 0, b 0.25 c 0.5, d 1 and e 2 mmol / l. While at a concentration of 2 mmol / l of calcium citrate in the F v solution over 16 hours, the F-V activity remained nearly constant, this fell at lower calcium concentrations from time-dependent.

From the foregoing, it is apparent that a concentration of calcium ions of at least 2 mmol Ca²⁺ / l in an environment of 10 mmol / l sodium citrate is sufficient to FV stabilize in a fresh gewonnnenen citrate, citrate-freezing plasma or kryoarmen citrate.

For the coagulation analysis mainly citrate is routinely used for its recovery upon presentation of a trisodium citrate blood is taken from a vein in the arm.

Such partially recalcified citrate is suitable as starting material for obtaining an F V concentrate.

If fresh human citrate (trisodium citrate final concentration: 11 mmol / l) was added 1 USP units / ml heparin and maintained this solution at 20 ° C, the F V activity was much more strongly than in the control without the addition of heparin. The addition of 5 mmol / L calcium chloride of this type of waste can also be drastically reduced even under these conditions.

This shows that citrate can be replaced more or less by heparin and the FV stabilized by calcium. So you can also achieve anticoagulant effect in this way, without revoking the FV that is necessary for its stabilizing calcium.

Calcium is not only a stabilization of FV in plasma possible but also in fractions prepared therefrom. In this case can be used as anticoagulants all those commonly used.

As F V-containing fraction that is useful as starting material for the production of a concentrate of the supernatant plasma can be used after separation of the cryoprecipitate. Also in the cryoprecipitate-free plasma can be stabilized by the FV calcium. This also applies to the operations, which can be used for obtaining an F V concentrate, for example, chromatography of a cryoprecipitate-free plasma on an anion exchanger.

F V-determination: The F V-determination can by the method of Koller, Loeliger and Duckert (Perlick, coagulation laboratory in clinics and practices, Publisher: VEB Georg Thieme, Leipzig, 1960) are performed.

For this purpose, 0.1 ml of F-V deficient plasma is mixed with 0.1 ml of a defined dilution of normal plasma. This mixture is incubated for 30 s at 37 ° C. Are then added in 0.2 ml of a calcium-containing thromboplastin, and the time until a clot appears. In this way, a reference curve is created.

In the quantitative determination of an F-V to be examined plasma concentrate or dilutions thereof are used in barbital buffer, instead of normal plasma in the test described above and determined the clotting time. Referring to a calibration curve obtained with a normal plasma dilution series, the V-F concentration can be determined.

One unit corresponds to the FV FV activity of 1 ml of normal plasma.

The following examples illustrate the invention.

example 1

F V stabilization in frozen plasma

Fresh human citrate plasma was prepared from 1 part won 110 mmol / L trisodium citrate and 9 parts whole blood by separating the blood cells. The plasma was frozen at -25 ° C and thawed after 24 hours, recalcified partiel and incubated at 37 ° C. At certain times of the F V activity was measured during incubation:<tables id="tabl0001" num="0001"><table frame="all"><title>Table 1</title><tgroup cols="4" colsep="1" rowsep="0"><colspec colnum="1" colname="col1" colwidth="39.37mm" /><colspec colnum="2" colname="col2" colwidth="39.37mm" /><colspec colnum="3" colname="col3" colwidth="39.37mm" /><colspec colnum="4" colname="col4" colwidth="39.37mm" /><thead valign="top"><row><entry namest="col1" nameend="col4" align="center">F V activity in thawed plasma was incubated without additives and 2.5 mmol or 5 mmol / l Ca²⁺.</entry></row><row><entry namest="col1" nameend="col1" align="center">incubation period</entry><entry namest="col2" nameend="col4" align="center">F V-residual activity in%</entry></row><row><entry namest="col1" nameend="col1" /><entry namest="col2" nameend="col2" align="center">Frozen plasma without additives</entry><entry namest="col3" nameend="col3" align="center">Frozen plasma + 2.5 mmol / l Ca²⁺</entry><entry namest="col4" nameend="col4" align="center">Frozen plasma + 5 mmol / l Ca²⁺</entry></row></thead><tbody valign="top"><row><entry namest="col1" nameend="col1" align="right">0</entry><entry namest="col2" nameend="col2" align="right">100</entry><entry namest="col3" nameend="col3" align="right">100</entry><entry namest="col4" nameend="col4" align="right">100</entry></row><row><entry namest="col1" nameend="col1" align="right">4 hrs.</entry><entry namest="col2" nameend="col2" align="right">59</entry><entry namest="col3" nameend="col3" align="right">81</entry><entry namest="col4" nameend="col4" align="right">81</entry></row><row><entry namest="col1" nameend="col1" align="right">6 hrs.</entry><entry namest="col2" nameend="col2" align="right">46</entry><entry namest="col3" nameend="col3" align="right">77</entry><entry namest="col4" nameend="col4" align="right">81</entry></row><row><entry namest="col1" nameend="col1" align="right">21 hrs.</entry><entry namest="col2" nameend="col2" align="right">10</entry><entry namest="col3" nameend="col3" align="right">38</entry><entry namest="col4" nameend="col4" align="right">74</entry></row></tbody></tgroup></table></tables>

example 2

F V-stabilization during recovery from a plasma fraction citrate

4.74 l of frozen human citrated plasma were thawed at 2-4 ° C and the undissolved residue, the cryoprecipitate is separated by centrifugation. To the supernatant 237 ml of a 1 g / 100 ml was used to remove the PPSB factors at 15 ° C containing Al (OH) ₃ suspension given, stirred for 15 minutes, the precipitate is separated by centrifugation and the solution containing 1 USP units / ml heparin added.

The solution was divided into 2 parts of each 2350 ml. Part 1 was treated with sufficient CaCl₂ that the concentration 5 mmol / L, and part 2 left that way. Both were processed under identical conditions to an F V concentrate, with the difference that contained for part 1 all solvents, namely Äquilibrier-, washing and dialysis media, 5 mmol / l CaCl₂.

To obtain the FV to any part of 32 g wet packed DEAE-Sephadex^(R)A 50, which had been equilibrated with a solution containing 60 mmol / l NaCl, 20 mmol / l trisodium citrate, and additionally for 1 part of 5 mmol / l CaCl₂ at pH 7.5, was added, and 1 h. stirred at 15 ° C.

The anion exchange resin was separated from the supernatant transferred to a column and washed free with 1.5 l physiological NaCl solution of plasma radicals.

Elution was carried out with a solution containing AT III and Part 2 further contained 1 mol / l NaCl, 1 USP-U / ml heparin 0.2 U / ml of 5 mmol / l CaCl₂. The eluate was collected in 3 fractions to 30 ml, 70 ml and 180 ml. Among the fractions 2 and 3 as much ammonium sulfate was added that 60% saturation was reached, the precipitate dissolved, and lyophilized in 5 ml physiological saline.

During the work-up with the addition of calcium chloride, the entire existing after aluminum hydroxide adsorption F V activity was recovered in the lyophilisate, were it only 20% without calcium.