CA1150607A - Process for the production of factor xa preparation - Google Patents
Process for the production of factor xa preparationInfo
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- CA1150607A CA1150607A CA000387534A CA387534A CA1150607A CA 1150607 A CA1150607 A CA 1150607A CA 000387534 A CA000387534 A CA 000387534A CA 387534 A CA387534 A CA 387534A CA 1150607 A CA1150607 A CA 1150607A
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- process according
- factor
- plasma
- activator
- prothrombin
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Abstract
ABSTRACT OF THE DISCLOSURE
A process is provided for the production of a Factor Xa preparation, comprising treating plasma with a prothrombin activator, removing suspended solids, mixing the thus obtained plasma liquid with a protein adsorbent to form a precipitate, eluting the precipitate with a protein elution agent and mixing the thus obtained eluate with-a Factor X activator, the Factor Xa pre-paration is employed in the determination of prothrombin for example, in the continuous monitoring of anticoagulation therapy.
A process is provided for the production of a Factor Xa preparation, comprising treating plasma with a prothrombin activator, removing suspended solids, mixing the thus obtained plasma liquid with a protein adsorbent to form a precipitate, eluting the precipitate with a protein elution agent and mixing the thus obtained eluate with-a Factor X activator, the Factor Xa pre-paration is employed in the determination of prothrombin for example, in the continuous monitoring of anticoagulation therapy.
Description
~5~3t~
The present invention is concerned with a process for producing Factor Xa which is employed in a reagent for the determination of prothrombin in biological material, for example, blood plasma.
This application is a divisional of Canadian Patent ~pplication, S.N. 318,481, filed December 2~, 197~.
The determination of the prothrombin level is an important clinical parameter for the continuous monitoring of anticoagulation therapy. Equally important is 1he detection of a Factor-II deficiency which can not only be inherited but can also be acquired, i.e., as a result of a primary funda-mental disease.
Starting from natural thrombin-substrate fibrinogen, in the course of the development of prothrombin determination methods, use is now made of synthetic chromogenic substrates, for example, peptide-E~nitroanilide derivatives, tissue thrombo-plastin being used as activator. One disadvantage of this method is, for example, the fact that the optimum activation time varies with the prothrombin concentration~ Furthermore, there is a lack of specificity. In the case of other sub-strates it would be necessary to have lower plasma concen-trations in the activation mixture in order to determine pro-thrombin in normal plasma. However, due to the high plasma dilution, it is not possible to achieve a complete activation in plasmas with low prothrombin activity.
Staphylocoagulase and snake venoms have also been used as activators. They activate prothrombin directly, however, it has been found that these venoms at least partly also activate the PIVKA-prothrombin which is formed during treatment with oral anticoagulants. Staphylocoagulase also reacts with the PIVKA-prothrombin.
Therefore, it is desirable to provide a process and a reagent for the determination of prothrombin in plasma which 6~7 permits the carrying out of a test with few sources of error, increased precision and sensitivity and improved reprod~cability.
Such a test is to be specific, that is to detect prothrombin but not PIVKA-prothrombin, All the prothrombin is to be con-verted quickly and completely into thrombin without the activator necessary for this purpose participating in any way in the colour reaction, There is disclosed a process for the determination of prothrombin in biological material, for example, in blood i~ plasma, by conversion o~ prothrombin into thrombin, enzymatic fission of a thrombin substrate with the thrombin and measure-ment of a fission product, wherein the test solution contain-ing the prothrombin is incubated with the addition o~ Factor Xa.
Although human Factor Xa has pro~ed to be best, it is, however, also possible to use, for example, bovine Factor Xa.
The principle of this method of determination can be explained as ~ollows: from, for example, oligopeptides in which E~nitroaniline is attached, as chromog~nic group, to the carboxyl group of arginine by amide formation, thrombin splits off E~nitroaniline,in a particular case this may be represented by the following:
N-Tos-Gly-pro-Arg-pNA thHrO~bin~
N-Tos-Gly-Pro-Arg-OH ~ pNA
Examples of thrombin substrates which have proved to be especially useful include N-~os-Gly-Pro-Arg-pNA and N-Cbz-Gly-Pro-Arg-pNA (Chromozyme TH*, Boehringer Mannheim Gmbh), as well as H-D-Phe-Pip-Arg-pNA (Substrate S-2238)** and Bz-Phe Val-Arg-pNA (Substrate S-2160)** The yellow colour of the free *trademark ** laboratory designation .
~-nitroaniline can be measured photometrically at abou~ 390 to 410 nm, the amount of coloured material liberated per unit time being proportional to the enzyme activity.
As buffer for carrying out the method, it is pre-ferred to use tris and/or imidazole buffer with a pH of about 8 to 9, to which hydrochloric acid and/or sodium chloride can be added~
Phospholipids and calcium chloride can be added as co-reagents for the activation of the prothrombin.
The addition of the substrate takes place after com~
plete conversion of the prothrombin into thrombin in the test solution, after the addition of Factor Xa.
The Factor Xa used is readily obtainable by a simple process which is the subject of the present invention.
;
According to this process, plasma and preferably human plasma is treatèd with a prothrom~in activator, suspended solids are removed, for example, by centrifugation, the resulting plasma liquid is mixed with a protein adsorbent, the precipitate obtained is eluted with a protein elution agent and the eluate obtained is mixed with a Factor X activator and optionally with a soluble calcium salt. The prothrombin acti-vator used is advantageously Echis carinatus venom but there can also be used other snake venoms, for example, Taipan snake venom (O~Yuranus scutellatus) and trypsin.
The coagulate of suspended solids obtained after treatment with the prothrombin activator, which is suitably centrifuged off, consists partly of fibrinogen, antithrombin and thrombin.
The protein adsorbent used is preferably barium sulphate, citrate or c~alate but there can also be used, for ~, . . .
~S~V7 example, aluminium hydroxide, DEAE-Sephadex* and QAE-Sephadex*
The protein elution agent used can be, for example, an aqueous solution of trisodium c:itrate, a phosphate buffer or an aqueous solution of sodium chloride.
Russell's viper venom is particularly preferred as the Factor X activator.
As soluble calcium salt, it is preferred to use calcium chloride.
If the eluate, after treatment with the protein elution agent, contains components which have a negative effect on the coagulation system, for example, calcium-binding com-ponents, for example, citrate ions when the protein elution agent is sodium citrate, it is necessary to introduce a dialysis step. In the case mentioned, for example, dialysis is carried out against a buffered physiological solution of sodium chloride (M~chaelis buffer) at a low temperature, i.e., at about 4C.
After the treatment with the Factor X activator and the addition of the calcium ion-containing solution, the pre-paration is, before use, left to stand at a low temperature and preferably at about 4C. for the complete conversion of Factor X into Factor Xa, Further steps can subsequently be carried out in order to obtain highly purified Factor Xa.
These further purification steps can comprise gel filtration with a molecular sieve which differentiates the range of from 50,000 to 100,000, for example Sephadex * G 100 or Biogel *
P 100, eluting with, for example, sodium acetate buffer (0~4 molar; pH 7). Furthermore, it is also possible to carry out an ammonium sulphate precipitation (for example, with a 45 to 55% solution pH 6 to 8), an ion exchange chromatography with DEAE-'Sephadex'* A 50, DEAE cellulose or QAE- Sephadex"*/
cellulose, using as buffer sodium potassium phosphate (for *trademark . ' example, 0.02 molar, pH 6.8) or a gradient of 0.1 to 1.0 molar aqueous sodium chloride solution. It is also possible to carry out a treatment with hydroxyapatite (phosphate buffer 0.2 to 0~5 molar; pH 6,8), as well as preparative electro-phoresis and possibly ultracentrifuging. The above-mentioned methods are advantageously combined. Factor Xa purified in this manner can be used in the test in the form of a solution, for example, in veronal or Michaelis buffer, in physiological sodium chloride solution or in the test buffer. However, for use in the test according to the present invention, this puri-fication procedure can be omitted and the Factor Xa pre-p æ ation, enriched in the above-described manner, used.
The present invention also provides a reagent for the determination of prothrombin, comprising a thrombin sub-strate and a prothrombin activator, the prothrombin activator being Factor Xa and preferably human Factor Xa~
This reagent preferably consists essentially of tris-and/or imidazole buffer and human Factor Xa as prothrombin activator. As co-reagents, it is advantageous to use phos~
pholipids from human brain and calcium chloride as well as a synthetic thrombin substrate.
It will be understood that the individual components of ~he reagent will be pre,sent in amounts to perform their intended function. Thus, for example, there should be sufficient thrombin substrate to react with all of the thrombin formed from the prothromhin, and Factor Xa should be employed in an amount effective to convert the prothrombin to thrombin.
Such a reagent preferably comprises:
0.3 to 6.5 ~g./ml. phospholipids, 0.7 to 10 m~M/litre calcium chloride and 150 to 380 ~M/litre substrate, ~5~)7 the Factor Xa concentration corresponding to 0.2 to 2.5%, by weight, of the plasma extract. The reagent can be in dried or dissoLved form.
The process and reagent for determination of pro-thrombin permit a rapid and dependable determination of pro-thrombin. The process is characterised by its specificity because Factor Xa detects normal prothrombin but not PIVKA-prothrombin. The previously employed activators certainly did not act specifically. The addition of a standardised Factor Xa preparation ensures that, in every case, a sufficient amount of activator is present. It was very surprising that Factor Xa, after the addition thereof, converted all of the pro-thrombin rapidly and completely into thrombin without itself ~ participating in any way in the colour reaction, which is of great importance. Thus, it is known that the Factor Xa has a direct splitting action on, for example, synthetic peptide-E~nitroanilide derivatives, for example, Chromozyme TH*. In particular, it was also surprising that human Factor Xa could be used more advantageously than, for example, bovine Factor Xa, It has proved to ~e especially advantageous to add Factor Xa in definite amounts; the activation of Factor Xa itself present in blood plasma would here give rise to unsatisfactory results. Furthermore, it was surprising that, by means of such a simple preparative process, such as that according to the present invention, an activator could be made available, which ensures practicability and especially technical useful-ness, and, having regard to its origin, namely human plasma, could be sufficiently p~oduced at all.
The fol:Lowing Examples are given for the purpose of illustrating the present invention:
*trademark Example 1 Production of a Factor Xa preparation .
Normal plasma is obtained from the blood of healthy donors with an average age of 30 years, half of the donors being male and half being female. The plasma contains 25 mM
sodium citrate. After a first centrifuging for 15 minutes at 1500 g and at ambient temperature, the plasma is subjected to a second centrifuging at 4C, for 30 minutes at 20,000 g.
Per 4 ml. normal plasma, there are added l ml. l/30 molar aqueous calcium chloride solution and 2 drops of Echis carinatus venom (Sigma V 8250* fundamental solutions in each case l mg./ml. water3 and left to stand for about 2 hours in a waterbath at 37C. The resulting coagulate is centrifuged o~f and 150 mg. barium sulphate is added to the supernatant obtained from each 4 ml. of normal plasma. The mixture is stirred for 30 minutes at ambient temperature and then centrifuged. The supernatant is discarded and the precipitate washed four times with approximately 4 ml. amounts of physio-logical sodium chloride solution. The centrifugate is eluted with 2 ml. of a 0.2 molar trisodium citrate solution (pH 7.0).
After again centrifuging, the supernatant is dialysed against physiological sodium chloride solution at 4C. The dialysate is stored in small amounts of about 250 ~l. at -20C, To each-portion is added, at ambient temperature, 30 ~l. of a 0.1 molar calcium chloride solution and l ul. Russell's viper venom (Vipera russelli, The Wellcome Foundation Limited) and the mixture is left to stand for about 14 hours at 4C. This preparation can be used in the test, 2 ~l. corresponding to 1% plasma extract. It c~n also be lyophilised. Further puri-fication can also be carried out in the manner described in detail hereinbefore.
*trademar]c 6C~7 Example 2 Method for the determination of prothrombin and thrombin, Test mixtures were prepared which contained the following components:
Substance ~ Volume Final Concentration buffer solution 400 ~1., minus the other amounts added test plasma 1 to 5 ~1. 0~25 to 1.25%
phospholipids 2 ~1~ 1.25 ~g./ml.
calcium chloride solution 40 ~1. (0.1 m) 10 mM/l.
activator ~
(Factor Xa pre- Corresponding paration accord- ¦ to 1% plasma ing to Example 1) 1 2 ~1. fraction The bu~fer solution is prepared as follows:
Stock solution A: O.lM/l. tris + 0.1 M/l imidazole 0.1 M/l. hydrochloric acid Stock solution B: 0.1 M/l. tris + 0.1 M/l. imidazole +
0~1 M/l. sodium chloride Stock solution C: 0.2 M/l. sodium chloride.
The buffer used (pH 8.4) is prepared by combining the stock solutions in the volume ratio'A:B:C of 1:1:2.
The phospholipids used were an acetone/diethyl ether extract of human brain obtained by Bell and Altonis method.
The components of the test mixture are pipetted directly in the given order into a microcuvette with a layer thickness of 1 cm. of an "Aminco DW 2"* spectrophotometer at 37C. The incubation time for Factor Xa is 120 seconds. There are then added th~ereto 50 ~1~ (1.5 mM/l.) substrate (Chromozyme TH*; Boehringer Mannheim GmbH or Substrate-2238**, Kabi), the end concentration thereby being 187.5 ~M/l. for Chromozyme TH*
*trademark ** laboratory designation 6~7 and 148 ~M/l. for Substrate-2238*, After thoroughly mixing as quickly as possible, commencement of measurement begins immediately.
The absorption per unit time is recorded directly with an indicator rate in minutes per cm. in the horizontal and an absorption in mm. per mm. full scale in the vertical.
Various absorption sensitivities and running speeds can hereby be selected. Therefore/ the change of absorption per minute can be caiculated according to the following formula:
measured absor~tion in mm x absorption sensitivity total scale ln mm -indicator rate in min./cm. x measurement stretch Example for measurement stretch 10 cm., vertical 43 mm., full scale 252 mm., absorption sensitivity 0.5 and running speed 0.033:
; 43 ~52 x 0.5 = 0.258 abSorption chanqe 0,033 x 10 min./cm mlnute *laboratory designation _ g _
The present invention is concerned with a process for producing Factor Xa which is employed in a reagent for the determination of prothrombin in biological material, for example, blood plasma.
This application is a divisional of Canadian Patent ~pplication, S.N. 318,481, filed December 2~, 197~.
The determination of the prothrombin level is an important clinical parameter for the continuous monitoring of anticoagulation therapy. Equally important is 1he detection of a Factor-II deficiency which can not only be inherited but can also be acquired, i.e., as a result of a primary funda-mental disease.
Starting from natural thrombin-substrate fibrinogen, in the course of the development of prothrombin determination methods, use is now made of synthetic chromogenic substrates, for example, peptide-E~nitroanilide derivatives, tissue thrombo-plastin being used as activator. One disadvantage of this method is, for example, the fact that the optimum activation time varies with the prothrombin concentration~ Furthermore, there is a lack of specificity. In the case of other sub-strates it would be necessary to have lower plasma concen-trations in the activation mixture in order to determine pro-thrombin in normal plasma. However, due to the high plasma dilution, it is not possible to achieve a complete activation in plasmas with low prothrombin activity.
Staphylocoagulase and snake venoms have also been used as activators. They activate prothrombin directly, however, it has been found that these venoms at least partly also activate the PIVKA-prothrombin which is formed during treatment with oral anticoagulants. Staphylocoagulase also reacts with the PIVKA-prothrombin.
Therefore, it is desirable to provide a process and a reagent for the determination of prothrombin in plasma which 6~7 permits the carrying out of a test with few sources of error, increased precision and sensitivity and improved reprod~cability.
Such a test is to be specific, that is to detect prothrombin but not PIVKA-prothrombin, All the prothrombin is to be con-verted quickly and completely into thrombin without the activator necessary for this purpose participating in any way in the colour reaction, There is disclosed a process for the determination of prothrombin in biological material, for example, in blood i~ plasma, by conversion o~ prothrombin into thrombin, enzymatic fission of a thrombin substrate with the thrombin and measure-ment of a fission product, wherein the test solution contain-ing the prothrombin is incubated with the addition o~ Factor Xa.
Although human Factor Xa has pro~ed to be best, it is, however, also possible to use, for example, bovine Factor Xa.
The principle of this method of determination can be explained as ~ollows: from, for example, oligopeptides in which E~nitroaniline is attached, as chromog~nic group, to the carboxyl group of arginine by amide formation, thrombin splits off E~nitroaniline,in a particular case this may be represented by the following:
N-Tos-Gly-pro-Arg-pNA thHrO~bin~
N-Tos-Gly-Pro-Arg-OH ~ pNA
Examples of thrombin substrates which have proved to be especially useful include N-~os-Gly-Pro-Arg-pNA and N-Cbz-Gly-Pro-Arg-pNA (Chromozyme TH*, Boehringer Mannheim Gmbh), as well as H-D-Phe-Pip-Arg-pNA (Substrate S-2238)** and Bz-Phe Val-Arg-pNA (Substrate S-2160)** The yellow colour of the free *trademark ** laboratory designation .
~-nitroaniline can be measured photometrically at abou~ 390 to 410 nm, the amount of coloured material liberated per unit time being proportional to the enzyme activity.
As buffer for carrying out the method, it is pre-ferred to use tris and/or imidazole buffer with a pH of about 8 to 9, to which hydrochloric acid and/or sodium chloride can be added~
Phospholipids and calcium chloride can be added as co-reagents for the activation of the prothrombin.
The addition of the substrate takes place after com~
plete conversion of the prothrombin into thrombin in the test solution, after the addition of Factor Xa.
The Factor Xa used is readily obtainable by a simple process which is the subject of the present invention.
;
According to this process, plasma and preferably human plasma is treatèd with a prothrom~in activator, suspended solids are removed, for example, by centrifugation, the resulting plasma liquid is mixed with a protein adsorbent, the precipitate obtained is eluted with a protein elution agent and the eluate obtained is mixed with a Factor X activator and optionally with a soluble calcium salt. The prothrombin acti-vator used is advantageously Echis carinatus venom but there can also be used other snake venoms, for example, Taipan snake venom (O~Yuranus scutellatus) and trypsin.
The coagulate of suspended solids obtained after treatment with the prothrombin activator, which is suitably centrifuged off, consists partly of fibrinogen, antithrombin and thrombin.
The protein adsorbent used is preferably barium sulphate, citrate or c~alate but there can also be used, for ~, . . .
~S~V7 example, aluminium hydroxide, DEAE-Sephadex* and QAE-Sephadex*
The protein elution agent used can be, for example, an aqueous solution of trisodium c:itrate, a phosphate buffer or an aqueous solution of sodium chloride.
Russell's viper venom is particularly preferred as the Factor X activator.
As soluble calcium salt, it is preferred to use calcium chloride.
If the eluate, after treatment with the protein elution agent, contains components which have a negative effect on the coagulation system, for example, calcium-binding com-ponents, for example, citrate ions when the protein elution agent is sodium citrate, it is necessary to introduce a dialysis step. In the case mentioned, for example, dialysis is carried out against a buffered physiological solution of sodium chloride (M~chaelis buffer) at a low temperature, i.e., at about 4C.
After the treatment with the Factor X activator and the addition of the calcium ion-containing solution, the pre-paration is, before use, left to stand at a low temperature and preferably at about 4C. for the complete conversion of Factor X into Factor Xa, Further steps can subsequently be carried out in order to obtain highly purified Factor Xa.
These further purification steps can comprise gel filtration with a molecular sieve which differentiates the range of from 50,000 to 100,000, for example Sephadex * G 100 or Biogel *
P 100, eluting with, for example, sodium acetate buffer (0~4 molar; pH 7). Furthermore, it is also possible to carry out an ammonium sulphate precipitation (for example, with a 45 to 55% solution pH 6 to 8), an ion exchange chromatography with DEAE-'Sephadex'* A 50, DEAE cellulose or QAE- Sephadex"*/
cellulose, using as buffer sodium potassium phosphate (for *trademark . ' example, 0.02 molar, pH 6.8) or a gradient of 0.1 to 1.0 molar aqueous sodium chloride solution. It is also possible to carry out a treatment with hydroxyapatite (phosphate buffer 0.2 to 0~5 molar; pH 6,8), as well as preparative electro-phoresis and possibly ultracentrifuging. The above-mentioned methods are advantageously combined. Factor Xa purified in this manner can be used in the test in the form of a solution, for example, in veronal or Michaelis buffer, in physiological sodium chloride solution or in the test buffer. However, for use in the test according to the present invention, this puri-fication procedure can be omitted and the Factor Xa pre-p æ ation, enriched in the above-described manner, used.
The present invention also provides a reagent for the determination of prothrombin, comprising a thrombin sub-strate and a prothrombin activator, the prothrombin activator being Factor Xa and preferably human Factor Xa~
This reagent preferably consists essentially of tris-and/or imidazole buffer and human Factor Xa as prothrombin activator. As co-reagents, it is advantageous to use phos~
pholipids from human brain and calcium chloride as well as a synthetic thrombin substrate.
It will be understood that the individual components of ~he reagent will be pre,sent in amounts to perform their intended function. Thus, for example, there should be sufficient thrombin substrate to react with all of the thrombin formed from the prothromhin, and Factor Xa should be employed in an amount effective to convert the prothrombin to thrombin.
Such a reagent preferably comprises:
0.3 to 6.5 ~g./ml. phospholipids, 0.7 to 10 m~M/litre calcium chloride and 150 to 380 ~M/litre substrate, ~5~)7 the Factor Xa concentration corresponding to 0.2 to 2.5%, by weight, of the plasma extract. The reagent can be in dried or dissoLved form.
The process and reagent for determination of pro-thrombin permit a rapid and dependable determination of pro-thrombin. The process is characterised by its specificity because Factor Xa detects normal prothrombin but not PIVKA-prothrombin. The previously employed activators certainly did not act specifically. The addition of a standardised Factor Xa preparation ensures that, in every case, a sufficient amount of activator is present. It was very surprising that Factor Xa, after the addition thereof, converted all of the pro-thrombin rapidly and completely into thrombin without itself ~ participating in any way in the colour reaction, which is of great importance. Thus, it is known that the Factor Xa has a direct splitting action on, for example, synthetic peptide-E~nitroanilide derivatives, for example, Chromozyme TH*. In particular, it was also surprising that human Factor Xa could be used more advantageously than, for example, bovine Factor Xa, It has proved to ~e especially advantageous to add Factor Xa in definite amounts; the activation of Factor Xa itself present in blood plasma would here give rise to unsatisfactory results. Furthermore, it was surprising that, by means of such a simple preparative process, such as that according to the present invention, an activator could be made available, which ensures practicability and especially technical useful-ness, and, having regard to its origin, namely human plasma, could be sufficiently p~oduced at all.
The fol:Lowing Examples are given for the purpose of illustrating the present invention:
*trademark Example 1 Production of a Factor Xa preparation .
Normal plasma is obtained from the blood of healthy donors with an average age of 30 years, half of the donors being male and half being female. The plasma contains 25 mM
sodium citrate. After a first centrifuging for 15 minutes at 1500 g and at ambient temperature, the plasma is subjected to a second centrifuging at 4C, for 30 minutes at 20,000 g.
Per 4 ml. normal plasma, there are added l ml. l/30 molar aqueous calcium chloride solution and 2 drops of Echis carinatus venom (Sigma V 8250* fundamental solutions in each case l mg./ml. water3 and left to stand for about 2 hours in a waterbath at 37C. The resulting coagulate is centrifuged o~f and 150 mg. barium sulphate is added to the supernatant obtained from each 4 ml. of normal plasma. The mixture is stirred for 30 minutes at ambient temperature and then centrifuged. The supernatant is discarded and the precipitate washed four times with approximately 4 ml. amounts of physio-logical sodium chloride solution. The centrifugate is eluted with 2 ml. of a 0.2 molar trisodium citrate solution (pH 7.0).
After again centrifuging, the supernatant is dialysed against physiological sodium chloride solution at 4C. The dialysate is stored in small amounts of about 250 ~l. at -20C, To each-portion is added, at ambient temperature, 30 ~l. of a 0.1 molar calcium chloride solution and l ul. Russell's viper venom (Vipera russelli, The Wellcome Foundation Limited) and the mixture is left to stand for about 14 hours at 4C. This preparation can be used in the test, 2 ~l. corresponding to 1% plasma extract. It c~n also be lyophilised. Further puri-fication can also be carried out in the manner described in detail hereinbefore.
*trademar]c 6C~7 Example 2 Method for the determination of prothrombin and thrombin, Test mixtures were prepared which contained the following components:
Substance ~ Volume Final Concentration buffer solution 400 ~1., minus the other amounts added test plasma 1 to 5 ~1. 0~25 to 1.25%
phospholipids 2 ~1~ 1.25 ~g./ml.
calcium chloride solution 40 ~1. (0.1 m) 10 mM/l.
activator ~
(Factor Xa pre- Corresponding paration accord- ¦ to 1% plasma ing to Example 1) 1 2 ~1. fraction The bu~fer solution is prepared as follows:
Stock solution A: O.lM/l. tris + 0.1 M/l imidazole 0.1 M/l. hydrochloric acid Stock solution B: 0.1 M/l. tris + 0.1 M/l. imidazole +
0~1 M/l. sodium chloride Stock solution C: 0.2 M/l. sodium chloride.
The buffer used (pH 8.4) is prepared by combining the stock solutions in the volume ratio'A:B:C of 1:1:2.
The phospholipids used were an acetone/diethyl ether extract of human brain obtained by Bell and Altonis method.
The components of the test mixture are pipetted directly in the given order into a microcuvette with a layer thickness of 1 cm. of an "Aminco DW 2"* spectrophotometer at 37C. The incubation time for Factor Xa is 120 seconds. There are then added th~ereto 50 ~1~ (1.5 mM/l.) substrate (Chromozyme TH*; Boehringer Mannheim GmbH or Substrate-2238**, Kabi), the end concentration thereby being 187.5 ~M/l. for Chromozyme TH*
*trademark ** laboratory designation 6~7 and 148 ~M/l. for Substrate-2238*, After thoroughly mixing as quickly as possible, commencement of measurement begins immediately.
The absorption per unit time is recorded directly with an indicator rate in minutes per cm. in the horizontal and an absorption in mm. per mm. full scale in the vertical.
Various absorption sensitivities and running speeds can hereby be selected. Therefore/ the change of absorption per minute can be caiculated according to the following formula:
measured absor~tion in mm x absorption sensitivity total scale ln mm -indicator rate in min./cm. x measurement stretch Example for measurement stretch 10 cm., vertical 43 mm., full scale 252 mm., absorption sensitivity 0.5 and running speed 0.033:
; 43 ~52 x 0.5 = 0.258 abSorption chanqe 0,033 x 10 min./cm mlnute *laboratory designation _ g _
Claims (13)
1. A process for the production of a Factor Xa preparation, comprising treating plasma with a prothrombin activator, removing suspended solids, mixing the thus obtained plasma liquid with a protein adsorbent to form a precipitate, eluting the precipitate with a protein elution agent and mixing the thus obtained eluate with a Factor X activator.
2. A process according to claim 1, wherein said eluate is mixed with said Factor X activator and a soluble calcium salt.
3. A process according to claim 1, wherein said sus-pended solids are removed by centrifuging.
4. A process according to claim 2, wherein said sus-pended solids are removed by centrifuging.
5. A process according to claim 1, wherein said plasma is human plasma.
6. A process according to claim 2, 3 or 4, wherein said plasma is human plasma.
7. A process according to claim 1, 2 or 5, wherein the prothrombin activator is Echis Carinatus venom.
8. A process according to claim 1, 2 or 5, wherein the protein adsorbent is barium sulphate.
9. A process according to claim 1, 2 or 5, wherein the protein elution agent is an aqueous sodium citrate solution.
10. A process according to claim 1, 2 or 5, wherein the Factor X activator is Russell's viper venom.
11. A process according to claim 2 or 4, wherein the soluble calcium salt is calcium chloride.
12. A process according to claim 1, 2 or 5, wherein the elution is carried out with a protein elution agent which contains calcium-binding components and the eluate is dialysed.
13. A process according to claim 1, 2 or 5, wherein further purification of the Factor Xa preparation is carried out by gel filtration with at least one of a molecular sieve, an ammonium sulphate precipitation, an ion exchange chromatography, preparative electrophoresis and ultracentrifuging.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA000387534A CA1150607A (en) | 1977-12-24 | 1981-10-07 | Process for the production of factor xa preparation |
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE19772757992 DE2757992A1 (en) | 1977-12-24 | 1977-12-24 | METHOD FOR PROTHROMBIN DETERMINATION |
| DEP2757992.6 | 1977-12-24 | ||
| CA318,481A CA1127942A (en) | 1977-12-24 | 1978-12-22 | Process and reagent for determination of prothrombin |
| CA000387534A CA1150607A (en) | 1977-12-24 | 1981-10-07 | Process for the production of factor xa preparation |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1150607A true CA1150607A (en) | 1983-07-26 |
Family
ID=27166024
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000387534A Expired CA1150607A (en) | 1977-12-24 | 1981-10-07 | Process for the production of factor xa preparation |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA1150607A (en) |
-
1981
- 1981-10-07 CA CA000387534A patent/CA1150607A/en not_active Expired
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