Process for producing a factor V concentrate.

The invention relates to a method for producing a factor V concentrate from human blood plasma or a fraction from blood plasma which contain an anticoagulant.

The vessels, platelets and plasmatic factors contribute to the biological phenomenon of hemostasis. The primary wound closure results from the synergistic interaction of vessels and platelets, which lead to the so-called primary wound closure, which is then solidified by the plasma coagulation, which leads to the formation of fibrin. The platelets form the reaction surface for the course of the plasma coagulation, in which 13 plasma factors, which inhibitors are not included, participate. These plasmatic factors are proteins, mainly glycoproteins, which are mainly serine proteases in their effect. These circulate in the form of inactive precursors in the blood and are activated in response to stimuli such as injuries. Activation is based on the principle of limited proteolysis, with the factor preceding in the coagulation cascade activating the successive factor following the principle of an enzyme-substrate reaction. Essential for the activation are phospholipids, as they are found in the form of a mixture, the so-called platelet factor 3 (PF 3), and calcium ions. In addition, so-called accelerators are important, proteins that are able to accelerate the coagulation process. Factors V and VIII (FV and F VIII) are among the accelerators that work in conjunction with phospholipids and calcium by forming so-called activation complexes.

FV is a ß-globulin with a molecular weight of 330,000 daltons, which is activated by a limited proteolysis of thrombin and by protein C_a is deactivated. This creates specific activation peptides.

The effect of FV as an accelerator is due to the fact that it intensifies the interaction between FX a and prothrombin in the presence of calcium and platelet phospholipids in such a way that thrombin formation is accelerated about 1000 times. The complex of the factors X a, phospholipids, calcium and FV a is called a prothrombin activator or prothrombinase. In accordance with the accelerator effect of the FV, a reduction in this coagulation factor leads to a slowdown in thrombin formation and thus to coagulation and, as a biological consequence, to an increased risk of bleeding.

All coagulation factors, regardless of their enzymatic character, are comparatively very unstable and sensitive to denaturation, at least in the form that is usually obtained from anticoagulated plasma. This applies in particular to the F VIII and the FV, the two accelerator proteins.

The instability of factor V is used in the production of F V-deficient plasma: human citrate plasma, which is incubated in a water bath at 37 ° C, loses its F V activity within 24 hours, while all other factors remain stable .

Congenital and acquired F V deficiency states are relatively rare. Nevertheless, there are a number of indications where an F V concentrate appears to be indicated. Due to the instability and denaturability, such a concentrate is not available to this day. This is in complete contrast to the other coagulation factors, which are all available in the form of concentrates for therapy. This illustrates the particular instability of this coagulation factor, which is reflected in the term "labile factor" for factor V.

For these reasons, there has been a need for a method for obtaining FV from whole blood that ensures the stability of this factor during the collection, 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 coagulation analysis and 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 production of factor V concentrate.

Although the FV is easily denaturable, it has already been isolated from plasma in an active form. This was possible by adding synthetic protease inhibitors to fresh citrate plasma, from which the prothrombin factors had been removed, as protection against enzymatic degradation of the FV. Such additives are not suitable for the production of a standard plasma and also for the purification of FV, since they are toxic and block the interaction of the FV with other coagulation factors.

It is also known to obtain factor V by chromatography on an anion exchanger from plasma, from which the cryoprecipitate has been separated. The disadvantage of this process is the low yield.

Surprisingly, it has been found that the loss of factor V activity when obtained from plasma is a function of the citrate concentration when citrate is used as an anticoagulant. While FV is relatively stable at 20 ° and 5 mmol / 1 citrate, denaturation occurs with increasing citrate concentrations. A citrate concentration of 5 mmol / 1 could not be undercut if coagulation should be avoided.

Surprisingly, it was also found that a loss of activity can be reduced by adding divalent cations, preferably calcium ions. While the F V activity in citrated plasma, which usually contains tri-Na citrate in a concentration of 11 mmol / 1, drops to values ​​close to 0% of the norm at 37 ° C. after 21 hours, 35% of the activity is retained if such a solution 2.5 mmol / 1 calcium ions, and 70% if it contains 5 mmol / 1 calcium ions.

The invention relates to a method for producing a concentrate of coagulation factor FV from a human blood plasma containing an anticoagulant or a fraction from human blood plasma containing an anticoagulant, characterized in that such a plasma or such a fraction is added to a salt of a divalent metal in an amount adds that a concentration of divalent cations is reached, which is less than the concentration equivalent to the concentration of anticoagulant, and obtains an F V concentrate in a known manner.

Ca, Mg or Mn are suitable as divalent metals.

Such a metal salt is preferably a soluble salt of calcium, especially calcium chloride or acetate. The concentration of divalent cations is preferably at least 1 mmol / l, particularly preferably greater than 2 mmol / 1. The effect does not have to increase steadily with concentration, but can go through a maximum. The concentration must of course not be so great that the mixture coagulates.

Anticoagulants are primarily salts of citric acid or heparin.

The calcium need not be added immediately after the blood is drawn. An advantageous effect is also achieved if calcium is added up to 12 hours after the blood has been drawn, especially if the plasma has been stored at 3-5 ° C. for so long. Adding calcium to frozen and thawed plasma or to the supernatant after separation of cryoprecipitate also significantly stabilizes the F V.

1 shows the dependence of the F V activity on the time and the calcium concentration. As in citrated plasma, the pH was 7.5-7.8, the temperature 4 ° C.

For this purpose, human citrate freezing plasma, which was obtained by freezing a mixture of 9 parts by volume whole blood and one part by volume sodium citrate and with a final citrate concentration of 10 mmol / 1, was thawed and against a physiological buffer with a citrate concentration of 10 mmol / 1 and one Calcium concentration of 0.25, 0.5, 1 or 2 mmol / 1 dialyzed.

In Fig. 1, a corresponds to a concentration of calcium of 0, b of 0.25, c of 0.5, d of 1 and e of 2 mmol / 1. While at a concentration of 2 mmol / 1 calcium in the citrate-containing F V solution the F V activity remained almost constant over 16 hours, it decreased over time with lower calcium concentrations.

From the foregoing it can be seen that a concentration of calcium ions of at least 2 mmol Ca 2+ / 1 in a milieu of 10 mmol / 1 sodium citrate is sufficient to stabilize the FV in a freshly obtained citrate plasma, a citrate freeze plasma or a low-cryocitrate plasma .

For the coagulation analysis, mainly citrate plasma is routinely used, for the extraction of which blood is drawn from an arm vein using a tri-Na citrate solution.

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

When fresh human citrate plasma (tri-Na citrate final concentration: 11 mmol / l) 1 USP-U / ml heparin was added and this solution was kept at 20 ° C., the F V activity decreased significantly more than in the control without added heparin. By adding 5 mmol / 1 calcium chloride, this drop in activity can be drastically reduced even under these conditions.

This shows that citrate can be replaced more or less with heparin and the FV can be stabilized with calcium. An anticoagulant effect can also be achieved in this way without depriving the FV of the calcium necessary for its stabilization.

Calcium not only stabilizes FV in plasma, but also in fractions made from it. All the commonly used anticoagulants can be used.

After the cryoprecipitate has been separated off, the plasma supernatant can be used as the F V-containing fraction which can be used as a starting material for the production of a concentrate. Calcium can also stabilize FV in cryoprecipitate-free plasma. This also applies to the work steps that can be used to obtain an F V concentrate, for example chromatography of a cryoprecipitate-free plasma on an anion exchanger.

F V determination: The F V determination can be carried out according to the method of Koller, Loeliger and Duckert (Perlick, coagulation laboratory in clinic and practice, publisher: VEB Georg Thieme, Leipzig, 1960).

For this purpose, 0.1 ml F V deficient plasma is mixed with 0.1 ml of a defined dilution of normal plasma. This mixture is incubated at 37 ° C for 30 sec. Then add 0.2 ml of a calcium-containing thromboplastin and determine the time until a clot occurs. In this way, a reference curve is created.

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

One unit of FV corresponds to the F V 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 obtained from 1 part 110 mmol / 1 tri-Na citrate and 9 parts whole blood by separating the blood cells. The plasma was frozen at -25 ° C and thawed again after 24 hours, partially recalcified and incubated at 37 ° C. F V activity was measured at certain times during the incubation:<tables id="tabl0001" num="0001"><img file="EP0244834A2_D0001.tif" /></tables>

Example 2

F V stabilization during the extraction of a plasma fraction from citrated plasma

4.74 l of frozen human citrate plasma were thawed at 2-4 ° C. and the undissolved residue, the cryoprecipitate, was separated off by centrifugation. To remove the PPSB factors, 237 ml of an Al (OH) containing 1 g / 100 ml was removed at 15 ° C.₃-Suspension given, stirred for 15 min, the precipitate separated by centrifugation and the solution mixed with 1 USP-U / ml heparin.

The solution was divided into 2 parts of 2350 ml each. Part 1 was made with so much CaC1₂ added that the concentration was 5 mmol / 1, and leave part 2 so. Both were processed under identical conditions to an F V concentrate, with the difference that for part 1 all solvents, namely equilibration, washing and dialysis media, 5 mmol / 1 CaCl₂ contained.

To obtain the FV, 32 g of moist-packed DEAE Sephadex (^R) A 50 with a solution containing 60 mmol / 1 NaCl, 20 mmol / 1 tri-Na citrate and for Part 1 an additional 5 mmol / 1 CaC1₂ contained, had been equilibrated at pH 7.5, added and stirred at 15 ° C. for 1 hour.

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

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

While in the work-up with the addition of calcium chloride the entire F V activity present after the aluminum hydroxide adsorption was found in the lyophilisate, without calcium it was only 20% of it.