Prothrombin Time Determining Reagent and Methods of Using and Preparing the Same

The invention relates to a reagent for the determination of the prothrombin time using a chromogenic substrate for thrombin.

The determination of the prothrombin time (PT) is one of the most frequently performed coagulation tests. Due to the use of the PT to monitor oral anticoagulant therapy with coumarin derivatives, this test has become of increasing importance, especially due to the fact that the success of therapy is crucially dependent on monitoring for maintenance of the therapeutic range.

In the determination of the PT, the plasma or blood sample is activated with a tissue thromboplastin from animal or human tissue. The tissue thromboplastin activates the factor VII in the exogenous coagulation system, and this factor liberates, via activation of factor X, thrombin from prothrombin. In conservative coagulation analyses, the thrombin is then determined by measurement of the coagulation time using the natural substrate fibrinogen. It is of crucial importance that the tissue thromboplastin has high sensitivity to all vitamin K-dependent coagulation factors of the exogenous coagulation pathway, II, VII and X, since these are decreased by the coumarin therapy. The British comparative thromboplastin, which is accepted as international standard, detects these factors with high sensitivity.

So-called II, VII and X reagents are also used for monitoring anticoagulant therapy. Factor V and fibrinogen are added to these thromboplastin reagents in order to rule out variations in the content of these factors. By this means, it becomes possible for plasma or blood samples to be stored for a certain time before being investigated, since factor V, which has a crucial effect on the PT, rapidly loses activity.

Since factor VII has the shortest biological halflife, it responds most rapidly to therapy with coumarin derivatives. For this reason, factor VII-sensitive thromboplastins are regarded as particularly suitable also for monitoring the initial phase of anticoagulation of a patient. Checks on factor X are likewise indicated in the initial phase (Aiach, M. et al., 1982, Thromb. Res. 47, 69-71), so that factor X-sensitivity is also desirable.

It has been possible for some time, due to the development of low molecular weight chromogenic peptide substrates for the activated factors of the coagulation cascade, to replace the classic coagulation substrate fibrinogen. Thus, nowadays there are relatively specific substrates for the vitamin K-dependent factors X and II. Sensitive methods for the determination of factors X, VII and II in plasma have been developed, and it has been possible to achieve relatively favorable correlations between the PT determination and the determinations of the individual factors. The disadvantage of all these determinations of the individual factors is that they detect only one of the vitamin K-dependent factors and thus do not reflect the pathophysiological situation of the patient in whom there is a decrease of all three factors. In particular, the critical factor VII cannot even nowadays be measured directly with a specific chromogenic substrate, but can only be measured via factor X.

A method has been described, in European Pat. No. A-0,014,039, in which the PT is determined using a chromogenic substrate. However, in order to achieve reasonable measurement times, a so-called serum reagent is needed, and this is obtained from recalcified plasma by thromboplastin activation. Experience has shown that the processing of the serum which is described does not guarantee the complete removal of all factors, in particular those of the exogenous coagulation pathway. Thus, a thromboplastin which is used in combination with a serum which has been prepared in the manner described cannot have a high factor-sensitivity, especially not when, as proposed in the patent, thrombin is added directly to the reagent.

Another method and reagent for the measurement of the prothrombin time using a chromogenic substrate have been presented in German Pat. No. A-3,113,350. In order to obtain a sufficiently stable reagent, it is necessary to use a specially prepared thromboplastin. This is because the thrombin substrate used in this application is subject to non-specific hydrolysis by proteases which are already present in the thromboplastin. However, these proteases were essentially removed during the modified method of preparation of the thromboplastin, so that the reagent has considerably improved stability. It is desirable, however, for reasons of comparability of photometric with coagulometric methods for the determination of the prothrombin time, for the two types of reagent to be prepared from the same thromboplastin. In addition, it is then possible to obtain a reagent for both photometry and coagulometry from one processing of thromboplastin.

Surprisingly, a reagent of this type can be prepared using conventional thromboplastins if a chromogenic substrate having high specificity for thrombin is used, for example H-D-Phe-Pro-Arg-ANBA-isopropylamide or other ANBA derivatives, such as ANBA-glycine ethyl ester or ANBA-neopentylamide. The invention relates to a reagent of this type.

Table 1 shows the results of hydrolysis of chromogenic substrates for various proteases by thromboplastin preparations of the state of the art. By measurement of the extinction at 405 nm, it is seen that thrombin substrates having the structure H-D-Phe-Pro-Arg-ANBA-R, Tos-Gly-Pro-Arg-ANBA-R or Boc-Gly-Pro-Arg-ANBA-R are hydrolyzed to only an inconsiderable extent by the thromboplastins, while a rapid consumption of the substrate is recorded for most of the other substrates including the substrate for thrombin used in German Pat. No. 3,113,350 A1, Tos-Gly-Pro-Arg-pNA. Thus all the thromboplastins exhibit a high proteolytic activity. This means in practice that this type of reagent with a substrate such as Tos-Gly-Pro-Arg-pNA is stable for only a very short time, since there is a rapid decrease in the amount of substrate which is used. In contrast, a reagent with, for example, H-D-Phe-Pro-Arg-ANBA-isopropylamide is stable for a considerably longer time and its stability is limited by the stability of the thromboplastin and not by the hydrolysis of the substrate.

Thus the properties of this type of reagent are very comparable with those of a coagulation reagent using the same thromboplastin.

Hence the invention relates to a reagent for the photometric determination of the prothrombin time, containing, at the least, a thromboplastin having proteolytic activity and a chromogenic substrate for thrombin, which comprises the chromogenic substrate for thrombin being converted by the thromboplastin having proteolytic activity, in aqueous suspension in the presence of calcium ions, to the extent of less than 0.00125 times the initial concentration per hour at 37.degree. C.

The thromboplastin is preferably obtained from human placenta.

A chromogenic substrate which is preferably used has the structure Tos-Gly-Pro-Arg-ANBA-R or H-D-Phe-Pro-Arg-ANBA-R, where R is NH--C.sub.n H.sub.2n+1, with n=1 to 7, NH-benzyl or the residue of an amino acid, or its alkyl ester, which is bonded via the .alpha.-amino group.

The reagent described can contain calcium ions at a concentration of 0.5 to 10 mmol/l, preferably 5 mmol/l.

It can additionally contain a buffer solution such as tris, HEPES, glycylglycine, EPPS and imidazole, preferably HEPES, at a concentration of 0.005 to 0.1 mol/l, preferably 0.025 mol/l, with a pH range from 7 to 8.5, preferably 7.6.

It is also possible to add neutral salts, preferably sodium chloride, at a concentration of 0.01 to 0.2 mol/l, preferably 0.05 mol/l.

This type of reagent can also contain a heparin inhibitor such as protamine salts or polybrene.RTM., particularly Polybrene.RTM., in a concentration of 0.01 to 0.5 mg/l, preferably 0.25 mg/l. Polybrene.RTM. is the trademark for Hexadimethrine bromide (1,5-dimethyl-1,5-diazaundecamethylen-polymethobromide).

It is also possible to add human or animal factor V to the reagent.

With the use of a plasma deficient in factors II, VII, X and V, the reagent describbed can be used for the determination of factors II, VII, X and V, or with the use of a plasma deficient in factors II, VII and X, it can be used for the determination of factors II, VII and X.

The abbreviations used are explained below:

ANBA: 5-amino-24-nitrobenzoic acid

Phe: phenylalanine

Pro: proline

Arg: arginine

Tos: toluenesulfonyl

Gly: glycine

Tris: trishydroxymethylaminomethane

HEPES: N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid

EPPS: 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid

Leu: leucine

pNA: para-nitroaniline

Suc: succinyl

Ala: alanine.

Difference in extinction after 92 hours at room temperature of a reagent prepared according to Example 1. The substrate concentration was 50 .mu.mol/l, and the thromboplastins were dissolved in accordance with the statements of the manufacturer and added at a concentration of 5%.

The examples which follow are intended to illustrate the invention further:

4 to 20 ml of human thromboplastin were added to 70 ml of an aqueous solution containing 5.844 g of NaCl, 1.47 g of CaCl.sub.2, 11.92 g of HEPES, 250 .mu.g of polybrene.RTM. and 67.1 mg of H-D-Phe-Pro-Arg-ANBA-isopropylamide HCl in one liter, pH 7.6, and the mixture was freeze-dried.

Depending on the amount and activity of the thromboplastin added, after reconstitution with 200 ml of water the time obtained for a change of 0.1 in extinction at 405 nm was from 20 to 40 sec with a normal plasma.

1. Reference curve

Reagent mixture as in Example 1. Serial dilutions of plasma with physiological sodium chloride solution were made up and the prothrombin time was measured by photometry.

500 .mu.l of reagent were added to 50 .mu.l of plasma or diluted plasma in a prewarmed plastic cell, and the extinction at 405 nm was recorded as a function of time. The time at which an increase of 0.1 in extinction is reached is determined from the graph. This time is plotted against the reciprocal of the plasma concentration. A straight line between 100 and 5% of normal is obtained, similar to the coagulation method. The normal figure for the undiluted plasma is 29.4 seconds. The times are considerably longer for pathological plasmas.

2. Factor sensitivity

The sensitivity of a reagent according to Example 1 for factors II, VII, X and V is shown in Table 2. Plasma was diluted with the appropriately deficient plasmas and analyzed as in 1.

It can be seen from Table 2 that the reagent responds sensitively to a decrease in the factors listed.