IE58463B1 - A procedure for the photometric determination of the activated partial thromboplastin time and a reagent for this purpose - Google Patents

Abstract

The method entails the use of a chromogenic substrate.

Description

The invention relates to a procedure for the photometric determination of the activated partial thromboplastin time (APTT) and to a reagent suitable for this purpose.

Determination of the activated partial thromboplastin time (APTT) is, in addition to the prothrombin time (thromboplastin time, Quick value), the test of coagulation which is carried out most frequently. The APTT permits conclusions to be drawn about the behavior of the endogenous pathway of coagulation. Another important use of this test is for monitoring heparin therapy. The APTT is sensitive to high molecular weight kininogen, prekallikrein and factors XII, X, IX, VIII, V and I, as well as to the inhibitors of coagulation, especially antithrombin III under heparin therapy and fibrinogen cleavage products (antithrombin VI), all of which prolong the APTT.

Reagents for the determination of the APTT essentially contain phospholipids and a suitable activator of the contact phase. By contact activation, factor XII is activated, and this then activates factor XI and prekallikrein. Due to the lipids and calcium ions contained in the reagent, activation of the entire endogenous pathway then occurs, and this terminates in formation of a fibrin clot. The time required for this to occur is the variable measured.

Inorganic materials are employed as activators of the contact phase, preferably celite or kaolin. Ellagic acid is also used (U.S. Patent 3,486,981 and German Offenlegungsschrfit 2,915,310). As an optically transparent reagent, ellagic acid has advantages for use in optical measuring procedures for detecting the formation of the clot. However, according to Bock et al., Biochemistry 20, 7258-7266 (1982), the active species is a complex of metal ions and ellagic acid, which is insoluble in water. Another activator is dextran sulfate.

However, these activators of the contact phase are non-physiologica I and are also difficult to standardize.

For example, the coagulation time of a kaο I in-activated APTT depends to a quite considerable extent on the particle size of the activator. Moreover, some coagulation factors are so strongly adsorbed on surface-active materials of this type, that the latter are used as adsorbents for the preparation of the contact factors. However, in addition, the composition of the phospholipid contained in a reagent of this type is essential for the result. Furthermore, the length of the preincubation time, after which the actual reaction leading to the formation of the clot is started by recalcification, plays sn important part, since activated coagulation factors are inhibited by inhibitors in the plasma, especially antithrombin III.

Sulfatides are regarded as being physiological activators of coagulation (Fujikawa et al., Biochem. 1 9 (1930), 1322-1330, and Tans and Griffin, Blood, 59 (1982), 69-75). Sulfatides belong to the class of g lycosphingolipids and they contain a sulfate group on the galactose ring. Various species belong to this group of I compounds, and they differ in the nature of the fatty acid chain. They can be detected in all tissues, in the membranes, and in especially large amounts in the brain, from which they can be obtained in a very pure form.

Sulfatides are more effective than kaolin for the contact activation of plasma.

The end point of conventional determinations of APTT is the measurement of a fibrin clot. Following the formation cf a clot of this type is possible only with IO technical difficulty. Quite a number of devices have been developed, and these make use of a variety of mechanical, electrical or optical procedures.

Since the introduction of chromogenic substrates for coagulation factors, attempts have also been made to employ them for the determination of the coagulation enzymes. The advantage of chromogenic substrates is the possibility of straightforward standardisation of the low molecular weight substrates, in contrast to the complex, high molecular weight natural substrates. The problem of measuring the formation of a clot no longer arises.

Moreover, chromogenic substrates have already been employed for carrying out global tests. Thus, Yair.ada and Meguro, Thrombos. Res. 15 (1979), 351-358, describe a method for measuring the APTT. This is based on activa25 tion of the endogenous pathway using ellagic acid in the presence of phospholipid, calcium and the chromogenic thrombin substrate H-D-Phe-Pip-Arg-pNA (p 2238). The disadvantages of this procedure are the use of a nonphysio logica I activator, the lack of specificity of the chromogenic substrate, and the extremely long measurement times (normal figure about 7.4 minutes). Only incomplete data has been published on the sensitivity to the individual clotting factors.

Another method is described by P. Aiyappa, Ann.

New York Acad. Sci. ¢1981), pages 812-821. In this technique of measurement, plasma is activated by ellagic acid in the presence of phospholipid and calcium ions for 5 minutes, and the thrombin formed in this time is measured using a chromogenic substrate. This method can lead to the formation of fibrin, and active thrombin undergoes inclusion in this. Moreover, thrombin can be further deactivated by inhibition. On the other hand, it is possible that, within the selected incubation time, the activation of pathological plasmas takes place either not at all or only incompletely, although they are actually capable of this, even though slowly. The procedure described by Aiyappa cannot be used to measure coagulation in deficient plasmas.

It has now been found, surprisingly, that it is possible to eliminate the essential disadvantages of the described APTT methods with chromogenic substrates by using a sulfatide as the physiological activator in combination with a highly specific thrombin substrate.

Thus the invention relates to a procedure for the determination of the activated partial thromboplastin time using an activator, a coagulation-active phospholipid or mixture of phospholipids, calcium ions and a chromogenic substrate for thrombin, which comprises the activator being a sulfatide or a mixture of sulfatides.

The invention also relates to a reagent for the determination of activated partial thromboplastin time, comprising a sulfatide, a phospholipid, a soluble calcium salt and a chromogenic substrate in the freeze-dri.ed form.

Utilisable sulfatides are commercially available, for example from Serva, Sigma or Supelco. It is advantageous to use sulfatides which have, on thin-layer chromatography on silica gel, an Rp of 0.2-0.3, preferably 0.25, with a mixture of chloroform/methanol/water 65:25:4 as the mobile phase and an Rp of 0.25-0.35, preferably 0.31, using chloroform/methanol 40:15. However, it is also possible to obtain sulfatides of this type by processes which have been described, for example by Hara and Radin, Anal. Biochem. 100, 364-370 (1979), or from dried acetone extract of brain. Worthwhile test concentrations are between 0.1 and 50 jjg/ml. The sulfatide can initially be suspended at a concentration of 0.01 g/l in 50 mmol/l of HEPES buffer, pH 7.6, and this suspension used for preparing the reagent.

The phospholipids which can be used are the animal and vegetable lipids employed in conventional reagents. It is particularly advantageous to use Lipids from human platelets or an extract from human placenta. It is important that these lipids have no thromboplastic activity, so that the exogenous pathway is not also activated.

The chromogenic substrate for thrombin must be specific for this enzyme to be possible to employ it in this test, since, of course, the intention is to measure thrombin selectively in the presence cf the other coagulation factors which are likewise activated. Chromozym* * Trade Mark ΤΗ (Tos-GIy-Prο-Arg-ρΝA ) and S 2160 (B z-P h e-V a I-A r g-pN A > are relatively non-specific and also indicate other coagulation factors, such as kallikrein or factors Xa and Xlla.

S 2238 (HD-Phe-Pip-Arg-pNA) is more suitable, although its specificity for the contact factors is again not very high. Apart from para-nitroanilides, peptides having other chromo phores are also suitable, for example, coumarin derivatives where the hydrolysis is followed by measuring the fluorescence.

The most suitable chromogenic peptides for a global test, such as APTT, are thrombin substrates such as are des cribed in German Patent Application 3,244,030.8. The chromophoric derivative they contain is 5-anino-2-nitrobe η z o i c acid.

A compound of the general formula I X-Pro-Arg-NH- (I) where R is C-j_5-alkyl or -CH CCH (CHj) £3 COOCH3, and X is Η-D-Phe-, Boc- Gly- or Tosyl-Gly-, is preferably used. The substrate is preferably employed at very low concentration. The best results are obtained with jjmol/l when H-D-Phe-Pro-Arg-ANBA-isopropy Iamide is used. The calcium concentration should be between 1 and mmol/l preferably 5 mmol/l.

The sulfatide, phospholipid, chromogenic substrate and the calcium salt, preferably calcium chloride, are dissolved or suspended at pH 7.2-8.5 in a buffer, such as HEPES or Tris. Imidazole, glycylglycine or triethanolamine buffers can also be used.

The procedure according to the invention is advantageously carried out at 25-37°C by mixing a plasma sample with the reagent at this temperature in a thermostated cell. The reaction is followed at 405-410 nm in a photometer.

With a typical normal plasma the extinction remains constant for some time and then increases greatly within a few seconds. The variable measured is the time necessary to reach a specified difference in extinction, for example 0.1. In principle, kinetic evaluation of the exponential curve is also possible. The reaction of a specified amount of substrate is analogous to that in a conventional APTT, where the activated thrombin reacts with a specified amount of fibrinogen and thus induces the clot. Example 1 shews that the new procedure in the preferred form indicates changes in the activity of each individual factor in the endogenous pathway.

In contrast to the convention APTT, in which, after a certain fixed activation time, the actual reaction leading to the clot is started by adding CaCl2, it is possible for CaCl2 to be added from the start in the new procedure, and this leads to there being one pipetting step less (monoreagent). However, in principle, a test system analogous to the procedure used hitherto is also possible with the procedure according to the invention. With this objective, the chromogenic peptide is added, together with CaCl2, after a specified preincubation time. In this case, it has proved to be favourable to add a little CaCl2 (about 1 mmol/l, to the sulfatide reagent even during ΙΟ the preincubation time, and to start the actual reaction by addition of more CaClj solution. The optimal final concentration in this case is again 5 mmol/l of CaClg.

The use of sulfatides for the contact activation of plasma, combined with chromogenic substrates, has not hitherto been disclosed. Manufacturers of reagents recommend that the sulfatides be stored below 0°C. Freeze-drying sulfatides together with the other necessary constituents of the reagent does not lead to a stable product. The APTT is always drastically increased.

Surprisingly, a stable and effective reagent is obtained by addition of serum albumin, gelatin or degraded and chemi cally crosslinked collagen. A concentration of only about 0.1-1% allows a lyophilisate of high activity to be produced.

As with conventional reagents, it is possible, in combination with deficient plasma to determine the individual factors of the endogenous pathway. For this purpose, the plasma sample will be diluted in order to eliminate the effect of the other coagulation factors, or it is employed in an excess of the deficient plasma used.

Addition of aminoacids, especially glutamine, asparagine or glutamic acid, increases the sensitivity of the reagent to heparin.

Abbrevi at ions: ANDA 5-amino-2-nitrobenzoic acid Arg Arginine Gly G ly c i ne HEPES N-2-hydrcxyethylpiperazinyl-N'-2-ethanesulfoni c acid Abrreviations continued: Phe Pheny lalanine Pip Pipecolic acid Pro Proline Tos Toluenesulfonyl Tris Tris(hydroxymethyl)amincmethane The invention is illustrated by the Example which follows. il Example : Factor sensitivity of the new reagent Reagent: 0.1 ml of 3mmol/l H-D-Phe-Pro-Arg-ANDA-isopropy l.amide chromogenic substrate (S 82 107) 0.5 ml of Fibraccel R 1:1000 (coagulationactive homologous phospholipid complex, Behringwerke AG) 0.5 ml of sulfatide solution (supplied by Supelco; Rp 0.25 with CHClj/MeOH H2O 65:25:4 on silica gel) 0.01 g/l .0 ml of buffer (HEPES, NaCl, Ca2+: 25, 50, m m o I / I; pH 7.6, 0.25X HaemaccelR (German patent 1,118,792 and 1,153,134) Mixture: 100 ρI of plasma or deficient plasma (Behringwerke), congenital F VII-deficient plasma ml of reagent 37°C, measurement of the time until E4Q5 nm = 0.1.

Deficient plasma Seconds Factor II 1,200 Factor V 1,200 Factor VIII 378 Factor IX 498 Factor X 448 Factor XI 396 Factor XII 336 HMWK (high molecular weight kininogen) 396 Kallikrein 720 Pooled plasma 170

Claims (10)

1. Patent Claims

1. A method for the determination of the activated partial thromboplastin time (APTT) by means of a chromogenic substrate and activation by sulfatides or mixtures of sulfatides, wherein all the reagents, apart from the sample, necessary for carrying out the test are added in one step.

2. The method as claimed in claim 1, wherein the chromogenic substrate is a compound of the formula I: X-Pro-Arg-NH -NO. CO-NH-R (I) where R is C x - 3 -alkyl or -CH[CH(CH 3 ) 2 ]COOCH 3 , and X is H-D-Phe- or tosyl-Gly-.

3. A reagent for the determination of the APTT by means of a method as claimed in claim 1, which contains all the components necessary for carrying out the test (a sulfatide or mixture of sulfatides, phospholipid, calcium ions and a chromogenic thrombin substrate) in one solution.

4. A reagent as claimed in claim 3, which contains a buffer, preferably HEPES, of pH 7.2 - 8.5.

5. A reagent as claimed in claim 3, which additionally contains an amino acid, preferably glutamine, asparagine or glutamic acid.

6. A reagent as claimed in claim 3, which additionally contains serum albumin, gelatin or a degraded and crosslinked collagen.

7. A reagent as claimed in claim 3, which is in freeze-dried form.

8. The method as claimed in claim 1, wherein the sample is diluted with an appropriate deficient plasma for determination of single factors.

9. A method as claimed in claim 1, substantially as hereinbefore described and exemplified.

10. A reagent as claimed in claim 3, substantially as hereinbefore described and exemplified.