DD250187A5 - Method for the photometric determination of protein C - Google Patents

Abstract

For the photometric determination of protein C, especially in plasma, the sample containing the protein C to be determined is incubated with thrombin to form activated protein C, then excess thrombin inhibitor, such as. B. antithrombin III, and then the decrease in the coagulation factors mediated formation of thrombin from prothrombin by means of a chromogenic thrombin substrate such. H-D-Phe-Pip-Arg-pNA or Tos-Gly-Pro-Arg-pNA. To reduce the formation of thrombin, an activator for factor XII, for example ellagic acid with the addition of kephatin, is added. As an activator for factor VII is z. B. thromboplastin and factor V and as an activator for factor II factor Xa added with the addition of kephatin. Fig. 1

Description

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Method for the photometric determination of protein G Field of application of the invention

The invention relates to a method for the photometric determination of protein C, in particular in plasma.

Protein C is a two-chain, vitamin K- dependent glycoprotein in plasma * Its synthesis takes place in the liver. Carboxylation of If-glutamic acid residues in the protein by a vitamin K-dependent carboxylase leads to the protein C. Protein C itself is a proenzyme and is converted by thrombin into activated protein C. In the first step, a precursor (decorboxy-protein C) is formed * The latter acts as an anticoagulant due to the inhibition of activated coagulation factors V and VIII »Decreased protein C levels in patients with liver disease» Consumption coagulopathy (DIC) and warfarin therapy «A congenital deficiency of protein C leads to venous thromboembolic risks« therefore, protein C plays an important role in both physiological Ha ^- mostasis and in many diseases, in particular in the Thrombosis * as a result, there is also a need for a simple, in particular photometric determination method for protein C. a specific chroraogenes protein C ~ substrate that's his direct photometric determination, but is not known.

Characteristic of the known state of the art

In the only commercially available test procedure

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The amount of protein C in the serum is determined by enzyme-labeled antibodies. However, this method has the disadvantage that the antibodies used for the determination also react with the above-mentioned decarboxy protein C. Since the serum concentration of decarboxy protein often rises sharply during treatment with anticoagulants , this procedure is a major source of error that may lead to inappropriate or inadequate therapy. R. B "Francis and M. D, Patch (Thrombosis Research 3_2, 605 613, 1983) teach a method for the determination of activated protein C in human plasma by determining the partial thromboplastin time (PTT test). In this case, the protein C is activated by the addition of thrombin and then the latter is inhibited by a deficiency of antithrombin III and heparin. "Heparin, in turn, is neutralized by a newly determined exact amount of protamine sulfate. Thereafter, protein C can be determined via the partial thromboplastin time.

The disadvantage of using immunological assay methods in plasma for protein C is that they do not provide information about the biological activity of the protein C molecules. The presence of abnormal protein C with greatly reduced biological activity (genetic variant) can not be found with such methods

R.M. Bertina et al (Thromb, Haemostas 5I ₁ (1) 1-5, (1984)) teach a spectrophotometric method for determining protein C activity. * This method comprises three independent steps:

1. Isolation of Protein C Using an Al (OH) Adsorption *

2. Activation of the plasma-separated protein G with thrombin and subsequent inhibition of the latter by equimolar amounts of antithrombin III and heparin,

3 · Measuring the proteolytic activity of isolated activated protein C with a chromogenic substrate = H pyro-Gly-Pro-Arg-pNA) ♦

This method is unsatisfactory in terms of specificity for protein C ₁ as this substrate is also cleaved by other clotting proteases. It can therefore lead to false positive results »

Aim of the invention ;

The aim of the invention is "to improve the accuracy of protein C determination,

Presentation of reading the invention

The invention has for its object to provide a simple and reliable optical test for the determination of active protein C available.

Activated protein C inactivates the coagulation factors Va and Villa, which in turn activate the production of thrombin from its non-active precursor, prothrombin.

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One could therefore think of determining active protein C via the formation or inhibition of thrombin formation from prothrombin. Since, however, protein C must first be activated by thrombin, but subsequently the activity of thrombin formed from prothrombin should be determined beforehand completely removed or inactivated for the activation of the protein added thrombin. In this inactivation, in order not to also inhibit the thrombin formed in the assay, the inhibitor would have to be dosed so accurately that it would be impractical to carry out a protein C assay according to the above-mentioned pattern for routine practice. "

It has now surprisingly been found that the added thrombin can be inactivated by an excess of antithrombin III without antithrombin III falsifying the measurement reactions »

The inventive method for the photometric determination of protein C, especially in plasma, is therefore characterized in that the sample containing the protein C to be incubated with thrombin to form activated protein C, then excess thrombin inhibitor is added and then the decrease of Coagulation factors mediated formation of thrombin from prothrombin is determined by means of a chromogenic thrombin substrate.

As a thrombin inhibitor, one of the known thrombin inhibitors such as antithrombin III (AT III), optionally

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Although suitable amounts used in the context of the invention as thrombin inhibitor AT III are generally dependent on the amount of thrombin present, since an excess of inhibitor must be used. In general, however, AT III levels between 0.5 and 100 U / ml, preferably 1 to 20 U / ml test volume are used. The inhibitor excess should generally amount to at least 10 %, preferably more than 20 %, based on the activity of throrabine. If, for example, 0.5 U thrombin is used for protein C activation, it is expedient to use 0.7 U or more after activation to thrombin inhibitor, in particular AT III.

Since activated protein C (APC) proteolytically inactivates the coagulation factors V and VIII, in the method according to the invention, the coagulation system is preferably modified by the addition of activators for coagulation factors and / or coagulation factors itself such that the inactivation of factors V and VIII in one possible pronounced reduction in thrombin formation. According to a first embodiment of the method according to the invention this is advantageously achieved by adding an activator for factor XII, for example ellagic acid with the addition of cephalin. According to a second embodiment of this preferred variant, an activator for factor VII, z, B, thromboplastin, and factor V are added. According to a third embodiment, factor Xa is added as an activator for factor II with the addition of cephalin.

These preferred embodiments of the invention take into account

Note the fact that the concentrations of the coagulation parameters Factors XII, XI, IX, VIII, X, V and II influence the time of thrombin generation in such a way that a certain thrombin threshold is reached the sooner the higher the concentration of these factors.

Thrombin formation is determined in the context of the invention by methods known per se. Suitable for this is the partial thromboplastin time (PTT) method. In its implementation, an activator for factor XII is expediently added. Thrombin formation can also be determined by the prothrombin time method. In this case it is expedient to add an activator for factor VII and F Va.

For the purposes of the invention, any chromogenic substrate suitable for determining thrombin can be used as the chromogenic thrombin substrate. However, H-D-Phe-Pip-Arg-pNA or Tos-Gly-Pro-Arg-pNA is preferably used in the context of the invention, where pNA = para-nitroaniline. The pNA is the chromogenic component of the substrates and is cleaved by the formed thrombin so that it can be determined photometrically in a known manner.

For example, a mixture of factors II to XII or substrate plasma (eg normal citrate plasma) can be used as coagulation system.

In the context of the invention, any plasma can be used, citrate plasma is preferred. Before protein C determination from plasma, sample preparation should be performed

become. Any adsorbent can be used which can bind vitamin K-dependent (carboxylated) proteins (eg, barium citrate, aluminum hydroxide). «

The process can be carried out at neutral or weakly alkaline pH values, preferably between pH 6 and 9. The buffer used may be the physiologically acceptable buffer active in this range, such as, for example, B »Tris / HCl. Furthermore, the stabilizers and preservatives customary for coagulation tests, such as bovine serum albumin, merthiolate and the like, can also be added.

AusfÖhrungsbeispiele

The following examples further illustrate the invention in conjunction with the accompanying drawings. In this represent:

FIG. 1: a calibration curve for the extension of the partial thromboplastin time by active protein C from plasma (150 to 0 %) and with Tos-Gly-Pro-Arg-pNA (ChromozymO TH) as chromogenic substrate.

Fig. 2: the extension of the partial thromboplastin time by pure active protein C with chromium as substrate.

FIG. 3: the extension of the prothroin time by pure active protein C with substrate Chromozyme © TH. FIG.

Fig. 4: the reduction of the initial slope by pure active protein C in a chrorogenic prothrombin test, substrate also chromozyme w TH.

example 1

Protein C determination from plasma by extension of the partial thromboplastin time (Factor VHI inactivation)

reagents:

Solution 1: 50 mmol / l sodium citrate _# 0.00025 % polyprene and 0.01 % merthiolate,

Solution 2: 250 mmol / l barium chloride, 150 mmol / l sodium chloride and 0.01 % merthiolate,

Solution 3: 200 mmol / 1 MES, 150 mmol / l sodium citrate and 0.01 % merthiolate, pH 6.0,

Solution 4 .: 6 U / ml thrombin in 100 mmol / l Tris / HCl, pH 8.0, 0.1 % BSA and 0.01 % Merthiolate,

Solution 5: 9 U / ml Antitrombin III and 0.25 USP / ml Heparin in 100 mmol / l Tris / HCl, pH 8, 100 mmol / l NaCl, 2 % BSA, 1.5 % sucrose and 0.01 % Merthiolate .

Reagent solution: cephalin and ellagic acid as factor XII-

Activator in 10 mmol / l Tris / HCl, pH 7.6,

Starting reagent: 1.1 mmol / 1 chromozyme ^ TH = Tos-Gly-Pro

Arg-pNA and 100 mbar / 1 calcium acetate,

a) Samples

Normal citrate plasma pool: Persons whose Quick-value is between 80 and 120 % are eligible as donors "

The calibration curve is created with the normal pool. The PC content of the normal citrate plasma pool is set to 100 % . Different protein concentrations are obtained by diluting the pool with sodium ditrate (20 mmol / l). Grades: 150 %, 100 %, 75%, 50 %, 25 %, 10 %, 0 % * For the 150% value, instead of 200, ul 300, μl pool is used as a sample.

Three citrated plasmas with different protein C antigen content are included as controls.

b) Sample preparation

200 Yul sample are incubated with 100 ul solution 1 for 10 minutes at 25 ⁰ C. After the addition of 200 μl of the ice-cold ( ⁰ ° C.) solution 2, it is incubated at ⁰ ° C. for 10 minutes. The sediment is centrifuged for 5 minutes at 4000 revolutions per minute (rotor diameter 17 cm) and OC, washed with 100 μl of the 1: 2 diluted with water 2 solution at ⁰ ° C. and centrifuged again. The sediment is taken up in 100 μl of solution 3 and incubated for 5 minutes at 25 ° C., finally centrifuged again. The supernatant = eluate also contains protein C in addition to other vitamin K-dependent proteins.

20 yul eluate (protein C) are mixed with 100 _/ ul solution 4 for 60 minutes at 37 ⁰ C incubated _e Protein C (PC) is active while PC (APC) is activated. To inactivate thrombin, 100 μl of solution 5 are then added and the mixture is incubated at 37 ° C. for 60 minutes. The mixture containing, in addition to activated protein C (APC), a

contains 50% excess of antithrombin III via thrombin, is used in the chromogenic coagulation test as a sample »

c) Measurement of the APC concentration by extending the partial thromboplastin time

50, Ul-activated protein C (mixture of b, see above), 100, μl of normal citrate plasma and 1000, μl of reagent solution

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are incubated in a microplastic cuvette for 5 minutes at 37.degree. C. After addition of 100 .mu.l of the starting reagent, the time is determined in a photometer at 405 nm and 37.degree. C. until a defined amount of substrate of newly formed thrombin is converted to Tos-Gly-Pro-Arg. Peptide and para-nitroaniline (= pNA) is implemented. The amount of reacted substrate being measured is defined by a threshold absorbance value (z * B * Λ E = 0.2). The concentration of coagulation parameters F XII, F XI, F IX, F VIII, FX, FV and F II are affected the time, the more concentrated they are, the faster the threshold is reached. "Since APC proteolytically inactivates factors VIII and V, the endogenous coagulation system (F XII-F II) is sensitively disrupted, thus prolonging the partial thromboplastin time (PTT). Under the given conditions, the prolongation of the partial thromboplastin time increases proportionally with the concentration of APC (or, PC in the plasma).

For evaluation, a calibration curve (FIG. 1) with the measured value pairs plasma / plasma dilutions (150 to 0 %) and PTT times is created. For plasmas of unknown PC concentrations

The PTT time is also determined and the corresponding PC concentration is read from the calibration curve.

d) Results

Table 1 summarizes the PTT times determined with plasma pool, plasma pool dilutions and control plasmas. The PTT times increase in proportion to the PC concentration in the sample (0 % to 150 %) . The protein C activities in the control plasma correspond to the protein C antigen concentrations, ie the recovery is good in the normal range (80 to 120 %) as in the abnormal range 20 to 40 %) .

Table 1 .

Prolongation of PTT time by APC from plasma dilutions and control plasmas

sample % Antigen PTT (see) % Activity 150 173 "5 150 plasma ioo 116 100 Dilute 75 108 75 calculations 50 93 50 25 76.5 25 10 68 10 O 60 O Kontroli- 114 121 102 plasrnen 81 115 84.5 23 _f 5 71 16

Example 2

Pure protein C - Determination by extension of the partial thromboplastin time (Factor VIII inactivation) The composition of the solutions is given in Example 1

a) Preparation

Protein C is purified from plasma according to the method of Comp et al., Blood 63 (1984) 15-21 * 20, μl of pure protein C in buffer, consisting of 100 mmol / l Tris / HCl, pH 8.0, 0 , 1% BSA and 0.01% merthiolate is incubated with 100 ml of solution 4 for 60 minutes at 37 ⁰ C _o thrombin is then inactivated by addition of 100, ul solution 5 and 60 minutes incubation at 37 C, "The activity of activated protein C in this mixture is determined with a substrate, Acetyl Pro-Pro-Arg-pNA (manufacturer: Pentapharm, Basel), whose para-nitroaniline is cleaved from APC (see * Lit * Bertina et al., Thromb, Haemostas (1) 1984, 1-5) and expressed in U / ml. 1 U / ml is defined as the amount of activated protein C contained in the mixture as described above for sample preparation of an iMormalcitratplasmapool (100 %) ,

b) Test procedure

20 μl of activated protein C (mixture of 2a, see above, different concentrations are obtained by dilution with buffer consisting of 100 mmol / l Tris / HCl, pH 8.0, 0.1 % BSA and 0.01 % merthiolate) , 100 μl plasma (composition: 5 parts factor VIII)

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Deficient plasma and 1 part of normal citrated plasma) and 100 μl of reagent solution are incubated in a microplastic cuvette for 5 minutes at 37 ° C. After addition of 100 μl start reagent, consisting of 1 * 1, umol / 1 chromozyme TH = Tos-Gly-Pro-Arg. pNA and 120, umol / 1 calcium lactate

in a photometer at 405 nm and 37 C determines the time until a defined amount of substrate of newly formed thrombin has been converted to Tos-Gly-Pro-Arg-peptide and para-nitroaniline. The amount of reacted substrate that is measured is defined by a threshold absorbance value (z, B ₄ Δ. £ = 0.2) »

c) Results

When the concentration of activated protein C in the mixture is varied from 0 to 10.6 U / ml, the time to reach the threshold (PTT time) increases from 96.6 to 744 seconds (FIG. 2). The increase in PTT time is directly proportional to 6.4 U / ml of the APC concentration.

Example 3

Protein C-3 Determination by Prolongation of Prothrornbin Time (Factor V Inactivation)

reagents:

Puffer solution: 100 mbar / 1 Tris / HCl, pH 8.0 and 0.1 % BSA

and 0.01 % merthiolate

Reagent solution: Rabbit brain thromboplastin as Factor VII activator, 1.7 μmol / 1 chromozyme

TH = los-Gly-Pro-Arg-pNA, 6 mmol / l CaCl ₂ in 100 mmol / l Tris / HCl, pH 8.1.

a) Preparation

It is also activated pure protein C with thrombin, then the thrombin with antithrombin III / heparin inactivated and used as a sample (see description 2a).

b) Test procedure

20 μl of activated protein C (mixture of 2a), 50 μl plasma (composition 1 part normal citrated plasma and 4 parts 0.9% NaCl solution) and 50 μl factor Va (IU / ml in buffer) are placed in a microplastic cuvette incubated for 10 minutes at 37 ^° C. * After addition of 1000 μl of reagent solution, the time is determined in a photometer at 405 nm and 37 ^° C. until a defined amount of substrate from newly formed thrombin to Tos-Gly-Prö-Arg peptide and para-nitroaniline is reacted. The amount of reacted substrate being measured is defined by a threshold absorbance value (eg AE = 0.1). The concentration of coagulation parameters F VII, FX, FV and F II affects the time - the higher the concentration, the more the threshold is reached faster. Since APC proteolytically inactivates factor V, the exogenous coagulation system (F VII - F II) is sensitively disturbed, thus prolonging the prothrombin time (PT). «

c) Results

Is the concentration of activated protein C in the

Varies from O to 10.6 U / ml, the time to reach the threshold (PT time) increases from 52.8 to 156 seconds (Fig. 3). The increase in PT _- Time is up to 6.4 U / ml of APC concentration directly proportional ·

Example 4

Protein C Determination by Chromogenic Prothrombin Assay "(Factor V Inactivation)

reagents:

Prothrombinreagenzlosung:

mmol / l Tris / imidazole / HCl, pH 8.4, 75 mmol / l NaCl, mmol / l CaCl "and 0.005 mmol / l cephalin,

a) Preparation

It is also activated pure protein C with thrombin, then inactivated the thrombin with antithrombin III / heparin and used as a sample (see description 2a),

b) Test procedure

20 μl of activated protein C (mixture of 2a), 50 μl plasma (composition 1 part normal citrated plasma and 60 parts 0.9% NaCl solution) as prothrombin and factor V source and 1000 / Ul prothrombin reagent are grown in a microplastic cuvette Incubated at 37 ⁰ C, after

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Addition of 20, ul factor Xa (2 U / ml in a 0.9%

NaCl solution) is incubated for a further 5 minutes at 37G. The color reaction is started (3 * 75 mmol / 1) with the addition of 50, ul substrate Chromozym TH and photometrically at 405 nm and 37 ⁰ C followed, "

The newly formed "thrombin converts the substrate to Tos-Gly-Pro-Arg-peptide and para-nitroaniline." The absorbance change per minute at the beginning of the reaction is measured. Under the conditions described, the concentrations of the coagulation factors Xa ₁ V and II influence the change in extinction Since APC proteolytically inactivates factor V, this system is susceptible to disruption, i.e., the conversion of prothrombin to thrombin has not been completed by the time of measurement and the more APC the lower the initial slope of the thrombin-substrate reaction present in the measuring approach,

c) Results

If the concentration of activated protein C in the mixture is increased from 0 to 10 _a 6 U / ml, the change in absorbance per minute decreases from 159 mU / min to 42 mE / min per minute (FIG. 4) APC concentration decreases exponentially.

Example 5

Influence of AT HI excess in the presence of heparin on the partial thromboplastin time - use of two thrombi

- 20 binsubstrate

a) Test procedure -. ,

50, μl of a mixture of AT III (concentration 0, 2.4, 3.6 / 4.9, 6.1 U / ml) and heparin as cofactor (concentration 0.12 USP / ml), 100, μl of normal citrated plasma and 1000, ul reagent according to Example 1 be

o incubated in a microplastic cuvette for 5 minutes at 37 ° C. After addition of 100, μl of starting reagent consisting of 1.1, umol / 1 substrate (Chromozym ^ iH = Tos-Gly-Pro-Arg-pNA or S 2238 = HD-Phe-Pip-ArgpNA) and 120, μl of calcium acetate, is in a photo

At 405 nm and 37 C, the time is determined until a defined amount of substrate of thrombin has been converted to the peptide and para-nitroaniline. The amount of reacted substrate that is measured is determined by a threshold absorbance value (eg Δ Ε = 0.2),

b) Results

Table 2 summarizes the PTT times determined in the absence and in the presence of AT III for both substrates. With both thrombin substrates, the PTT times are not prolonged by AT III addition, i. H. newly formed thrombin reacts with AT Ill-Oberschuß under the given reaction conditions, initially with the substrate »

Table 2

AT III influence on the PTT substrates: Chromozym * TH and S 2238

heparin AT III PTT (sec) " ^ TH PTT (see) USP / ml U / ml Chromozyme ^ P 2238 0.12 0 75 - 72.5 2.4 72.5 72.5 3.6 72 72 4.9 72 71 6.1 72 73

Claims (1)

claims 1 method for the photometric determination of protein C _# in particular in plasma, characterized in that the protein containing the protein C to be determined is incubated with thrombin to form activated protein C, then excess thrombin inhibitor is added and subsequently the decrease of mediated by coagulation factors Formation of thrombin from · prothrombin by means of a chromogenic thrombin substrate is determined. Verfahren » Process according to Claim 1, characterized in that antithrombin III, if appropriate with the addition of heparin, is used as the thrombin inhibitor, 3 »method according to claim 1 or 2, characterized in that is used as the chromogenic thrombin substrate H-D-Phe-Pip-Arg-pNA or Tos-Gly-Pro-Arg-pNA. 4 «Method according to one of claims 1 to 3, characterized in that an activator for factor XII is added wi ra · 5 _# Method according to claim 4, characterized in that cephalin and ellagic acid are added _e 6, Process according to Claims 1 to 3, characterized in that an activator for factor VII is added » -Z- 7, Method according to claim 6, characterized in that factor V and as Factor VII activator added thromboplastin v / earth. 8 * Process according to one of Claims 1 to 3, characterized in that an activator for factor II is added, 9 * Method according to claim 8, characterized in that cephalin and factor Xa are added « - 4 pages drawings -