DK162179B - REAGENTS FOR THE STEP DETERMINATION OF THE ACTIVATED PARTIAL THROMBOPLASTIN TIME AND THE USE OF THE REAGENT - Google Patents

Abstract

The method entails the use of a chromogenic substrate.

Description

DK 162179 B

The present invention relates to a reagent for one-step determination of the activated partial thromboplast time (APTT) and the use of this reagent.

The determination of the activated partial thromboplastin time (APTT) is next to the thromboplastin time (prothromic bone time, quick value) the most frequently performed coagulation test. APTT allows statements about the conditions surrounding the endogenous path of coagulation. A further important application of this test lies in the control of heparin therapy. The APTT comprises the high molecular weight kininogen, precallicrin and factors XII, X, IX, VIII, V and I, and also the inhibitors of the coagulation, in particular antithrombin III by the heparin therapy and the fibrinogen cleavage products (antithrombin VI), all of which prolong the APTT.

Reagents for the determination of APTT contain essentially phospholipids and a suitable "contact phase" activator. Upon contact activation, factor XII is activated, which then activates factor XI and precallikrein. By means of the lipids and calcium ions contained in the reagent, there is then provided an activation of the total endogenous pathway, which activation ends in a fibrin coagulate formation.

The time needed up to this point is the measurement parameter.

Inorganic materials are used as activators for the contact phase, preferably Celite or kaolin. In addition, ellagic acid is used, cf. U.S. Patent No. 3,486,981 and DE Publication No. 2,915,310. Ellagic acid, as an optically clear reagent, offers the advantages of using optical measurement methods to detect the coagulation formation. However, according to Bock et al., Biochemistry 20, 7258-7266 30 (1982), the active material must be a water-insoluble complex of metal ions and ellagic acid. An additional activator is dextran sulfate.

However, these contact phase activators are unphysiological and also difficult to standardize. For example, the coagulation time of a kaolin-activated APTT depends substantially on the particle size of the activator.

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Some coagulation factors are also adsorbed to the extent of such surfactants that they are used as adsorbents to prepare the contact factors. In addition, however, the composition of phospholipids containing such a reagent is also important for the result.

Also, the length of the pre-incubation period after which the actual reaction leading to coagulation formation is initiated by "recalcification" plays an important role, since activated coagulation factors are inhibited by the plasmatic inhibitors, especially antithrombin III.

Physiological activators for the coagulation include sulfatides, cf. Fujikawa et al., Biochem. 19, 1322-1330 (1980), as well as Tans and Griffin, Blood 59, 69-75 (1982). Sulfatides belong to compound class 15 of the glycosphingolipids and contain a sulfate group on the galactose ring. To this group of compounds belong different types which differ from each other by the nature of the fatty acid chain. They can be detected in all tissues of the membranes, in especially abundant amounts in the brain, from which they can be extracted in even extremely pure form. Sul-20 fatides are more effective than kaolin in contacting the plasma.

Conventional APTT best emulsifiers are based on the measurement of a fibrin clot. The formation of such a coagulate can, from a technical point of view, only be followed with difficulty. A variety of apparatus have been developed using various mechanical, electrical or optical methods.

Since the introduction of chromogenic substrates for coagulation factors, it has also been attempted to use these for the determination of coagulation enzymes. The advantage of chromogenic substrates lies in the simple standardizability of the low molecular weight substrates as opposed to the complex, high molecular weight natural substrates. The problem of measuring the formation of a coagulate lapses.

Also, chromogenic substrates have already been used for conducting "giobal tests". Thus, Yamada and Meguro describe Thrombos. Res. 15, 351-358 (1979), 3

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a method for implementing APTT. This method relies on the activation of the endogenous pathway with ellagic acid in the presence of phospholipid, calcium, and the chromogenic thrombin substrate H-D-Phe-Pip-Arg-pNA (S 2238). Disadvantages of this method are the use of an unphysiological activator, the lack of specificity of the chromogenic substrate and the extremely long measurement times (normal value about 7.4 minutes). As to the sensitivity of the single factors, only incomplete data have been published.

A further method is described by P. Aiyappa,

Ann. New York Acad.Sci., 812-821 (1981). In this measurement system, plasma with ellagic acid is activated in the presence of phospholipid and calcium ions for 5 minutes, and thrombin formed until then is measured with a chromogenic substrate. This method 15 can lead to the formation of fibrin which contains active thrombin. Thrombin can also be deactivated again by inhibition. On the other hand, within the selected incubation time, pathological plasmas may not activate at all or only incompletely, although they are actually capable of doing so, but with delay. The method described by Aiyappa does not allow measurement of a coagulation in deficiency plasmas.

It has now surprisingly been found that significant disadvantages of the described APTT methods with chromogenic substrates 25 can be overcome by using a sulfatide as a physiological activator in conjunction with a high specific thrombin substrate.

The invention thus relates to a reagent for one-step determination of the activated partial thromboplastin time, and this reagent is characterized in that it consists of a buffer, preferably HEPES, having a pH of 7.2-8.5, a sulfatide or a sulfatide mixture, a phospholipid, a soluble calcium salt and a chromogenic substrate for thrombin.

The invention further relates to the use of such a reagent in combination with a deficiency plasma from a factor of the endogenous pathway for determining this factor.

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Useful sulfatides are commercially available, e.g. from the companies Serva, Sigma or Supelco. Advantageously, such sulfatides are used which, by thin layer chromatography on silica gel and with a mixture of chloroform, methanol and 5: 65: 25: 4 as solvent, have an Rf value of 0.2-0.3, preferably 0.25 and using a mixture of chloroform and methanol in the ratio of 40:15 as the solvent has an Rf value of 0.25-0.35, preferably 0.31. However, such sulfatides can also be obtained using described methods, e.g. according to Hara and Radin, Anal. Biochem.

100, 364-370 (1979), or from brain acetone dry powder. Appropriate test concentrations are between 0.1 and 50 µg per day. ml. The sulfatide can first be suspended at a concentration of 0.01 g / liter in 50 mmol / liter of HEPES buffer, pH 7.6, and used for the preparation of the reagent.

As phospholipid, the animal and vegetable lipids used in conventional reagents may be used. Particularly advantageous is the use of lipids from human platelets or an extract of human placenta. Importantly, these 20 lipids do not exhibit any thromboplastic activity so that the exogenous pathway is also not activated.

The chromogenic substrate for thrombin must be specific to be used in this test, since the thrombin must be selectively measured alongside the other also activated coagulation factors. Chromoxym® TH (Tos-Gly-Pro - Arg-pNA) and S 2160 (Bz-Phe-Val-Arg-pNA) are relatively nonspecific and also indicate other coagulation factors such as kallikrein or factors Xa and XIIa. More suitable is s 2238 (HD-Phe-Pip-Arg-pNA), although in this case also the specificity of the contact factors is not very high.

In addition to para-nitroanilides, peptides with other chromophores are also useful, e.g. coumarin derivatives, the hydrolysis being followed by measuring the fluorescence.

The most suitable chromogenic peptides for a global test such as APTT are thrombin substrates such as these, e.g. is disclosed in DE Publication No. 3,244,030. The 5

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contains as chromophore derivatives of 5-amino ~ 2-nitrobenzoic acid.

Preferably, a compound of the general formula I is X-Pro-Arg-NH- <(jX-NO- \ Z2O-NH K (1> 10) wherein R is C1-C4 alkyl or -CH (CH (CH3) 2) COOCH3, and X is HD-Phe, BOC-Gly or Tosyl-Gly,

The substrate is preferably used at very low concentration. The best results are obtained at 50 μιηοΐ / liter using H-D-Phe-Pro-Arg-ANBS-isopropylamide. The calcium concentration should be between 1 and 10 mmol / liter and a concentration of 5 mmol / liter is preferred.

Sulfatide, phospholipid, chromogenic substrate and the calcium salt, preferably calcium chloride, are dissolved or suspended in a buffer such as HEPES or TRIS with a pH of 7.2-8.5. Imidazole, glycylglycine or triethanolamine buffers may also be used.

The present process is conveniently carried out at 25-37 ° C, comparing a plasma sample with the tempered reagent in a thermostatically controlled cuvette. The reaction is followed in a photometer at 405-410 nm.

In a typical normal plasma, the extinction remains constant for some time, then rapidly increases within a few seconds. The measurement parameter is the time required to reach a particular extinction difference, e.g. 0.1. In principle, a kinetic evaluation of the exponential curve is also possible. The reaction of a certain amount of substrate is analogous to the reaction of a conventional APTT, where the activated thrombin converts a certain amount of fibrinogen and thus triggers the coagulation. The following example shows that the present process in the preferred form shows changes in the activity of each factor in the

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6 endogenous pathway.

In contrast to the conventional APTT, where after a certain fixed activation time the actual, coagulation-leading reaction is initiated by CaCl 2 'addition, the CaCl (monoreagens). The chromogenic peptide is added together with CaCl2 only after a certain pre-incubation time. It has thereby been found to be advantageous also to add some CaCl2 to the sulfatide reagent (about 1 mmol CaCl2 per liter) during the pre-incubation time phase and to start the actual reaction by adding more CaCl2 solution. As a final concentration, here, too, 5 mmol CaCl liter optimally.

The use of sulfatides for the contact activation of plasma in combination with chromogenic substrates has so far been unknown. Regenerators recommend storage of the sulfatides at below 0 ° C. Lyophilization of sulfatides together with the necessary other constituents of the reagent does not lead to any stable product. APTT is still drastically extended.

Surprisingly, by the addition of serum albumin gelatin or a degraded or crosslinked collagen, a stable, active reagent is obtained. A concentration of just approx.

0.1 to approx. 1% allows the preparation of a good activity lyophilisate.

As with conventional reagents, in the absence of plasma, the determination of the single factors from the endogenous pathway is possible. For this purpose, the plasma sample 30 is diluted to exclude the influence of the other coagulation factors or the sample is used in deficit relative to the deficiency plasma used, as is the case with the use of conventional reagents, a quantitative determination.

An addition of amino acids, especially glutamine, aspartic acid or glutamic acid, increases the reagent's heparin sensitivity.

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Abbreviations: ANBS 5-Amino-2-nitrobenzoic acid

Arg Arginine 5 Gly Glycine HEPES N-2-hydroxyethylpiperazin-N'-2-ethanesulfonic acid

Phe Phenylalanine

Pip Pipecolinic acid

Pro Prolin 10 Tos Toluenesulfony1 TRIS Tris (hydroxymethyl) aminomethane

The following example serves to illustrate the invention.

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Example

Factor sensitivity for the nve reagent

Reagent: 0.1 ml 3 mmol / liter HD-Phe-Pro-Arg-ANBS isopropylamide chromogenic substrate (S 82 107) 0.5 ml Fibraccel® 1: 1000 (coagulation-active homologous phospholipid complex, Béhringwerke AG) 0 , 5 ml of sulfatide solution (Fa. Supelco; Rf = 0.25 with CHCl 3: MeOH: H 2 O = 65: 25: 4 on silica gel), 0.01 g / liter, and

5.0 ml buffer (HEPES, NaCl, Ca 2+: 25, 50, 5 mmol / liter; pH = 7.6, 0.25% Haemaccel® (DE

U.S. Patent Nos. 1,118,792;

1,153,134).

Starting material: 100 μΐ plasma or deficiency plasma (Behringwerke), F VII deficiency plasma congenitally, and 1 ml of reagent.

37 ° C, measuring time to E405nm = 0.1.

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Mana Plasma Seconds

Factor II 1,200

Factor V 1,200

Factor VII 207 5 Factor VIII 378

Factor IX 498

Factor X 448

Factor XI 396

Factor XII 336 10 HMWK (High Molecular Kininogen) 396

Kallikrein 720

Pool Plasma 170

Claims (4)

Reagent for one-step determination of the activated partial thromboplastin time, characterized in that it consists of a buffer, preferably HEPES, having a pH of 7.2-8.5, a sulfatide or sulfatide mixture, a phospholipid, a soluble calcium salt and a chromogenic substrate for thrombin. Reagent according to claim 1, characterized in that the chromogenic substrate is a compound of the general formula I X-Pro-Arg-NH -, - / - NX-NO, (X) \ M4-NH-R Wherein X represents Cx-C5 alkyl or -CH (CH (CH3) 2) COOCH3 and X represents HD-Phe-, BOC-Gly- or Tosyl-Gly-. Reagent according to claim 1, characterized in that it contains serum albumin gelatin or a degraded or crosslinked collagen. Use of a reagent according to any one of claims 1-3 in combination with a deficiency plasma from a factor of the endogenous pathway for determining this factor.