Use of annexins as a lupus anticoagulant control or standard in clotting tests

The invention relates to a process for the production of plasmas with added annexins for use as a control or standard in all functional clotting tests which are used for the detection of a lupus anticoagulant.

The invention relates to a process for the production of plasmas for use as
 a control or standard in all functional clotting tests which are used for
 the detection of a lupus anticoagulant.
 Lupus anticoagulants are immunoglobulins and belong to the acquired
 autoantibodies type. They are directed against phospholipids or
 phospholipid/protein complexes and prolong the clotting time in customary
 diagnostic clotting tests (see Triplett D., et al. Hematologic Pathology
 1988; 2: 121-143). These immunoglobulins are to be differentiated from
 other autoantibodies likewise against lipids, in particular cardiolipins.
 Both groups are clinically assigned to the antiphospholipid syndrome (APS)
 which manifests itself in thromboses and an increase in birth
 complications (miscarriages). The pathological mechanism of the lupus
 anticoagulants is still unclarified for several reasons. Firstly, the
 specificity of the occurring antibodies and thus the mechanism of action
 is individually different from patient to patient. Secondly, the
 subclasses of the immunoglobulins (IgM, IgG, IgA) and the antibody titers
 vary. Thirdly, there is a paradox between the determination of the lupus
 anticoagulant and clinical manifestation: a prolongation of the clotting
 time in in vitro tests, as is caused by lupus anticoagulant, points to an
 increased proneness to bleeding, in vivo, however, it is manifested in an
 increased proneness to thromboses.
 The diagnosis of a lupus anticoagulant is therefore restricted to a
 phenomenological, description of the behavior of a plasma sample in
 various clotting tests according to the recommendations of international
 committees (Brandt, J. T. et al., Thrombosis Haemostasis 1995; 74:
 1185-1190). These tests are the activated partial thromboplastin time
 (APTT), the kaolin clotting time (KCT) the dilute thromboplastin time
 (dPT) and the Russell's viper venom time (RVVT). A prolongation of the
 clotting time in these tests, however, is also obtained by a factor
 deficiency, which is why in the so-called plasma exchange test the
 pathological sample is mixed with normal plasma and as a rule determined
 in the APTT. A factor deficiency, as a rule, is already compensated for in
 a substitution of 50% by mixing with the normal plasma, while in the
 presence of a lupus anticoagulant pathological results are still obtained.
 Furthermore, the phospholipid dependence is to be checked, which is
 carried out using the same reagents, but with different concentrations of
 phospholipids. Furthermore to be differentiated are autoantibodies against
 individual clotting factors, which are likewise not compensated for by 1+1
 mixing with a normal plasma. As a rule, these factor antibodies, however,
 only act in one of the two pathways of the clotting system (in particular
 so-called Factor VIII inhibitors) and are recognized by the comparison of
 the various pathways, i.e. by the comparison of the abovementioned tests
 (APTT for the intrinsic, PT for the extrinsic pathway).
 The sensitivity of the reagents of the abovementioned tests to lupus
 anticoagulant is very different (Messmore, H., et al., Thrombosis and
 Hemostasis. 1994, 20: 79-94). Furthermore, lupus anticoagulants do not
 produce a pathological result in all tests, which is why the use of at
 least two functional tests is recommended (for example the APTT and the
 RVVT; see Brandt, J. T. et al.,Thrombosis Haemostasis 1995; 74:
 1185-1190). For comparison or exchange of data, e.g. in clinical studies,
 reference to a standard would therefore be useful, as well as regular
 checking of the test results, for example for monitoring a therapy. The
 use of individual plasma donors is unsuitable for the preparation of such
 a standard or a control for commercial use, however, because of the
 heterogeneity of the specificity, the low reproducibility and the poor
 stability of lupus anticoagulants. At present therefore, there is neither
 a clear determination of a lupus anticoagulant nor a reference to the
 quantification of a lupus anticoagulant.
 The invention was therefore based on. the object of finding a process with
 which, in a sample, e.g. plasma, a lupus anticoagulant can be reproducibly
 and quantifiably simulated in such a way that this modified plasma
 produces pathological results comparable with the presence of a natural
 lupus anticoagulant in all customary functional clotting tests and is thus
 suitable for quantification of the action (standard) and as a control.
 An essential characteristic of the lupus anticoagulant is the dependence of
 the clotting-prolonging property on the availability of the phospholipids.
 One possibility of simulating this behavior was described by Babcock and
 McGlasson (U.S. Pat. No. 4,877,741). They use an extract from a spider
 (Loxosceles reclusa) which contains an enzyme having a sphingomyelinase D
 activity. They were able to show (McGlasson, D. L. et al., Am. J. Clin.
 Pathol 1993; 100: 576-578) that this extract in the APTT leads with
 various reagents to a prolongation of the clotting time and can be
 neutralized by addition of phospholipids. This prolongation, however, is
 only weakly pronounced. Thus the prolongation achieved was at most 63% of
 the upper standard range (APTT reagent from Pacific Hemostasis; Table 2;
 (McGlasson, D. L. et al., Am. J. Clin. Pathol 1993; 100: 576-578). This is
 too low in order to produce typical prolongations of more than 100%, as
 occur with high lupus anticoagulant and therefore not adequate for
 standardization using.a wide measuring range.
 It has also been described that annexins under certain circumstances can
 lead to a prolongation of the APTT.
 This family of intracellular proteins at present includes at least eight
 characterized proteins which are designated according to. the new
 nomenclature as annexin I to VIII (Romisch, J. et al., Biol, Chem. Hoppe
 Seyler 1990; 5: 383-388). These proteins have an inflammation-modulating
 action in that they are released from cells on inflammation and bind to
 membrane surfaces and thereby inhibit the binding of phospholipase A2, an
 important step for the formation of arachidonic acid derivatives having an
 inflammatory activity. By means of this calcium-dependent binding to
 phospholipid surfaces, the clotting processes taking place on these
 surfaces are also disturbed, which is why these proteins are also
 designated as "vascular anticoagulant". Typically, the presence of
 annexins leads to a prolongation of the APTT in a concentration-dependent
 manner (Romisch et al. Thrombosis Research 1990; 60: 355-366).
 Until now, however, it was unknown whether annexins could fulfill all
 criteria comparably to a natural lupus anticoagulant. The following
 criteria are used for the functional diagnosis of a lupus anticoagulant
 (Brandt, J. T. et al., Thrombosis Haemostasis 1995; 74: 1185-1190):
 1. Prolongation of the clotting time beyond the normal range in at least
 two tests, in particular the APTT and the RVVT.
 2. Proof that this prolongation is phospholipid-dependent and can be
 neutralized either by addition of phospholipids or by use of the same
 reagents as in 1. only with higher phospholipid concentrations.
 3. Differentiation of a possible factor deficiency by plasma exchange
 experiments, in which case, in a 3+1, 1+1 and 1+3 mixture with a normal
 plasma in a clotting test, preferably the APTT, this prolongation is
 characteristically only neutralized by a high dilution (1+3), while in the
 case of a factor deficiency this is already clear earlier.
 4. Differentiation of an acquired inhibitor (auto antibody against a
 clotting factor), in particular against Factor IX or Factor VIII (acquired
 hemophilia A or B) wherein this only acts in one of the two clotting
 pathways (intrinsic or extrinsic pathway), while a lupus anticoagulant
 non-specifically affects all phospholipid-dependent stages in clotting
 tests.
 Surprisingly, it was now possible to show that by the suitable use of
 annexins a lupus anticoagulant standard or control preparation can be
 prepared which fulfills all the criteria described above.
 Example 1 shows the prolongation of the customary diagnostic clotting
 tests, the APTT, the PT and the RVVT on addition of an annexin (annexin V)
 to a plasma pool from normal blood donors (standard human plasma). With an
 addition of 125 .mu.g of annexin V, prolongations of the clotting times of
 470%, 31% and 200% above the upper normal range of the respective reagents
 were attained. This shows that, compared with the present state of the art
 (McGlasson, D. L. et al., Am. J. Clin. Pathol 1993; 100: 576-578) markedly
 higher prolongations of the clotting times and thus the possibility of the
 preparation of a standard and thus of reaching a further calibration range
 was improved. In the PT, this prolongation is not as strongly pronounced.
 In contrast to the APTT and RVVT, the PT is not recommended as a
 diagnostic test for lupus anticoagulants.
 Example 1 thus shows the fulfillment of criterion 1 of lupus diagnosis,
 i.e. a pathological result in at least 2 functional clotting tests, in
 particular the APTT and the RVVT. The two natural lupus
 anticoagulant-positive plasmas behave comparably.
 In Example 1, a plasma with factor deficiency, or a plasma with an acquired
 inhibitor, for example, were also tested. The deficiency of Factor VIII or
 of the acquired inhibitor against Factor VII only leads to pathological
 results in the APTT and not also, like the annexin-containing plasma or
 the natural lupus anticoagulant-positive plasmas, in the PT or the RVWT.
 Criterion 4 of lupus anticoagulant diagnosis is thus also fulfilled.
 Example 2 shows the results of the testing of phospholipid dependence in
 the APTT, the PT and the RVVT. Reagents having a phospholipid content
 which was modified compared with the reagents used in Example 1 were used.
 The ratio of the clotting time at low phospholipid concentration compared
 with that at high phospholipid concentration is increased in plasmas in
 which a lupus anticoagulant is suspected compared with normal plasmas.
 This behavior is only recorded in the required APTT and RVVT in the case
 of the natural and the simulated lupus anticoagulant-containing plasma.
 The PT is also pathological in this case. Thus criterion 2 of the lupus
 diagnosis is fulfilled. The normal plasma pool, as well as the
 factor-deficient plasma, appear to be nonpathological. Only the plasma
 with an acquired factor inhibitor remains prolonged in the process based
 on the APTT.
 Example 3 shows the results of testing for factor deficiency by means of
 plasma exchange experiments in the APTT. In the case of a mixture of 1+3
 with the normal plasma pool, all pathological plasmas, as well as the
 simulated lupus anticoagulant-positive plasma, are prolonged. In the case
 of a 1+1 dilution, the deficiency in the factor-deficient plasma is
 already adequately compensated. The clotting time is in the normal range.
 Annexin-containing plasmas, the natural, lupus anti-coagulant-positive
 plasmas, as well as the factor inhibitor-containing plasma are still
 pathological in the test. This shows, on the one hand, the fulfillment of
 the 3rd and last criterion for the diagnosis of a lupus anticoagulant and
 thus the possibility of employing annexins for the simulation of a lupus
 anticoagulant for use as calibrators or controls in functional clotting
 tests.
 Example 3 furthermore shows that for the fulfillment of the 3rd criterion,
 the plasma exchange experiment, the anticoagulatory active concentration
 of the annexin in a control must be adjusted such that in a 1+3 dilution
 with a normal plasma pool in an APTT a pathological result is still
 obtained. In the undiluted state, this corresponds in these examples to an
 addition of at least 7 mg/l of annexin V. Even in the case of this minimum
 amount, in the undiluted plasma a prolongation of the clotting time beyond
 the upper normal range of 87% is thus achieved (see Example 1, Table 1,
 APTT few PL). This requirement has not been possible by means of
 previously described processes (McGlasson, D. L. et al., Am. J. Clin.
 Pathol 1993; 100: 576-578) and thus it was not possible with plasmas
 prepared by this previous process to produce a lupus anticoagulant-like
 behavior in the plasma exchange experiment. The inventive process is thus
 clearly superior to the previous process.
 Apart from the functional clotting tests shown in the examples, the control
 or calibrator plasmas according to the invention can also be used in other
 functional clotting tests known to the person skilled in the art
 (Messmore, H. et al., Thrombosis Haemostasis 1994; 71: 220-224.; Brandt,
 J. T., Thrombosis Haemostasis 1991; 66: 453-458; Arnout, J. et al., Brit.
 J. Haematol. 1994; 87: 94-99; Rauch, J. et al., Thrombosis Haemostasis
 1989; 62: 892-896).
 Apart from the annexin V used in the examples, other proteins from the
 annexins family can also be used individually or as mixtures of various
 annexins for the preparation of control and/or calibrator plasmas.
 Advantageously, in this case both annexing purified from known natural
 sources (EP 0 123 307) or prepared by recombinant means (for example EP 0
 271 885) are used.
 In a control plasma, the concentration of these annexins is preferably
 selected such that even at a dilution of this control plasma of 1+3 with a
 normal plasma pool an APTT above the normal range is still obtained. In
 the case of adjustment of the control to other clotting tests, it is
 possible for these threshold concentrations to differ from one another in
 a reagent- and test specific manner.
 For calibrator plasmas, the concentrations of the annexins are ideally
 selected such that the clotting times obtained therewith are above the
 respective test and reagent used.
 From the use of defined amounts of annexins for production of a
 prolongation of the clotting time, the possibility results of the
 calibration of antibody titers of lupus anticoagulant-positive plasmas,
 for example, in mg/l of annexin equivalent.

A The following examples illustrate the invention.
 If not stated otherwise, reagents and plasmas from Behringwerke are used
 for the examples below.
 EXAMPLE 1
 Behavior of an annexin-containing plasma in diagnostic clotting tests in
 comparison with various normal and pathological plasmas.
 A normal plasma pool (standard human plasma) was mixed with 4 to 500 mg/l
 of recombinant annexin V (prepared according to EP 0 271 885) and the
 clotting times in the APTT (reagent: Pathromtin SL or Pathromtin SL with a
 10-fold higher phospholipid concentration than in pathromtin SL), the PT
 (reagent: Thromborel S) and the RVVT (regent: LA screen; manufacturer:
 Gradipore, Australia) on an automatic clotting apparatus (Amelung,
 Germany).
 Table 1 shows the prolongation of the customary diagnostic clotting tests,
 the APTT, the PT and the RVVT on addition of an annexin (annexin V) to a
 normal plasma pool. With an addition of 125 .mu.g of annexin V, the
 clotting times were prolonged by &gt;400%, 31% and 200% above the upper
 normal range of the respective reagents (APTT: 38 sec or 49 sec; PT: 13
 sec; RVVT: 45 sec; the instructions of the manufacturer).
 TABLE 1
 Effect of annexin V in a normal plasma pool (NPP) on
 the diagnostic clotting tests, APTT, PT and RVVT.
 APTT
 Content of annexin APTT [sec] [sec] PT [sec] RVVT [sec]
 V in NPP (in mg/l) many PL few PL many PL few PL
 0 38.0 45.5 13.0 38.8
 3.8 37.6 59.1
 7.3 38.8 91.6
 15.5 40.1 149.3
 31.3 345.0 186.3
 62.5 81.7 211.6 15.0 122.6
 125 215.9 262.5 17.0 135.1
 250 233.5 270.0 21.2 139.4
 500 320.6 316.0 34.5 152.8
 Furthermore, using these diagnostic tests the clotting times of the
 following pathological plasmas were determined:
 Factor VIII-deficient plasma (Prod. No. OTXW) for the simulation of a
 factor deficiency;
 a citrate plasma of a patient with acquired Factor VIII inhibitor (George
 King, USA);
 2 citrate plasmas of patients with proven lupus anticoagulant (Trina,
 Switzerland).
 Table 2 shows the results of determination of the APTT, the PT and the RVVT
 in the various plasmas. The normal plasma pool is within the normal range
 in all 3 tests. The Factor VIII-deficient plasma, however, is pathological
 in the APTT, as well as the plasma with an acquired Factor VIII inhibitor.
 Both plasmas are only slightly pathological in the PT, but nonpathological
 in the RVVT. In contrast to this, the two natural lupus
 anticoagulant-positive plasmas are pathological in all tests, even if
 marked to a differingly high extent. This nonspecific prolongation of the
 clotting times in the diagnostic clotting tests is produced in an
 identical manner by the addition of annexin to a normal plasma pool (the
 results from Table 1 with an addition of 125 mg/l or 62.5 mg/l of annexin
 were assumed in Table 2).
 TABLE 2
 Behavior of a normal plasma pool (NPP), a plasma with factor deficiency
 (FVIII-MP) or inhibitor against Factor VIII (anti-FVIII-P), natural lupus
 anticoagulant-positive plasmas (LA 1; LA 2) and a simulated lupus
 anticoagulant (NPP+annexin) in diagnostic clotting tests, APTT, PT and
 RVVT. The normal range of these tests with the reagents used is
 furthermore indicated.
 TABLE 2
 APTT RVVT
 Plasma sample [sec] PT [sec] [sec]
 NPP 38.0 13.0 38.8
 FVIII-MP 124.3 14.5 44.5
 anti-FVIII-P 133.6 13.5 38.0
 LA 1 56.5 14.5 136.3
 LA 2 55.4 14.5 133.3
 NPP + 63 mg/l annexin 81.7 15.0 122.6
 normal range 26-38 sec 10-13 sec 31-45 sec
 EXAMPLE 2
 Phospholipid dependence of the behavior of an annexin-containing plasma in
 diagnostic clotting tests in comparison with various normal and
 pathological plasmas.
 For the testing of the phospholipid dependence, the APTT, PT and RVVT of
 the pathological plasmas mentioned in Example 1, and of the normal plasma
 pool and of a simulated lupus anticoagulant control, consisting of normal
 plasma pool and 125 or 63 mg/ml of annexin V, was determined using
 reagents which, compared with Example 1, have another phospholipid
 content. For the APPT, this was the reagent Pathromtin SL with a
 phospholipid concentration of Pathromtin SL reduced by 90% compared with
 Example 1. For the PT determination, Thromborel S 1:50 was diluted with
 12.5 mM calcium chloride solution according to Brauer et al. (J Clin Chem
 Clin Biochem 1990; 28: 701). In contrast to this, for the RVVT, a
 lupus-sensitive reagent, i.e. with few phospholipids, is already used in
 the routine. For the negative control, in this example with LA confirm, a
 reagent having a higher phospholipid concentration was therefore employed.
 The results are shown in Table 3.
 TABLE 3
 Behavior of a normal plasma pool (NPP), a plasma with factor deficiency
 (FVIII-MP) or inhibitor against Factor VIII (anti-FVIII-P), natural lupus
 anti-coagulant-positive plasmas (LA 1; LA 2) and a simulated lupus
 anticoagulant (NPP+annexin) in diagnostic clotting tests, APTT, PT and
 RVVT, with altered phospholipid content (PL) compared with Example 1.

APTT [sec] PT [sec] RVVT [sec]
 Plasma sample PL reduced PL reduced PL increased
 MPP 45.5 21.5 34.0
 FVIII-MP 130.6 27.6 37.0
 Anti-FVIII-P 178.2 24.2 35.8
 LA 1 162.7 44.8 45.5
 LA 2 156.8 39.7 42.0
 NPP + 63 mg/l annexin 211.6 54.0 34.7
 normal range 32-49 sec 16-27 sec 34-51 sec
 Since in the presence of many phospholipids disturbance of the clotting by
 lupus anticoagulant is lowered, the quotient of the clotting time in the
 presence of few phospholipids to that in the presence of many
 phospholipids was formed from the clotting times obtained in Tables 2
 (Example 1) and 3 (see Table 4). A shortening of the clotting time on
 account of the neutralization of the inhibitory action of a lupus
 anticoagulant is expressed in an increased quotient. The permissible
 highest limits determined for these reagents, below which a lupus
 anticoagulant is not suspected, are shown in Table 4 for the reagents used
 here.
 It can be seen that the normal plasma pool, even in the presence of a
 factor deficiency, produces quotients in the normal range in all tests.
 The plasma with a specific inhibitor, on the other hand, is only still
 pathological in the APTT ratio. For the natural lupus
 anticoagulant-containing plasmas, as well as for the simulated lupus
 anticoagulant plasma according to the invention, a nonspecific reaction
 with phospholipids was otained in all 3 tests as a result of quotients
 which were clearly increased compared with the normal range.
 TABLE 4
 Calculation of the phospholipid dependence of a normal plasma pool (NPP), a
 plasma with factor deficiency (FVIII-MP) or inhibitor against Factor VIII
 (anti-FVIII-P), natural lupus anticoagulant-positive plasmas (LA 1; LA 2)
 and a simulated lupus anticoagulant (NPP+annexin) in diagnostic clotting
 tests, APTT, PT and RVVT, by formation of the quotient of the clotting
 time in the presence of few phospholipds to that in the case of many
 phospholipids. The permissible threshold value of the quotient, above
 which a lupus anticoagulant is suspected, is furthermore indicated.

Plasma sample APTT ratio PT ratio RVVT ratio
 NPP 1.2 1.7 1.1
 FVIII-MP 1.1 1.9 1.2
 anti-FVIII-P 1.3 1.8 1.1
 LA 1 2.9 3.1 3.0
 LA 2 2.8 2.7 3.2
 NPP + 63 mg/l annexin 2.6 3.6 3.5
 upper limiting value 1.2 2.1 1.3
 EXAMPLE 3
 Behavior of an annexin-containing plasma and various pathological plasmas
 in the plasma exchange experiment.
 The plasma exchange experiment -was carried out in the APTT using
 Pathromtin SL. For this purpose, the patho-logical plasmas mentioned in
 Example 1 as well as the simulated lupus anticoagulant-positive plasma
 3+1, 1+1 and 1+3 were mixed with a normal plasma pool and the APTT was
 determined. The results are shown in Table 5.
 TABLE 5
 Behavior of a plasma with factor deficiency (FVIII-MP) or inhibitor against
 Factor VIII (anti-FVIII-P), natural lupus anticoagulant-positive plasmas
 (LA 1; LA 2) and a simulated lupus anticoagulant (NPP+annexin) in the
 plasma exchange experiment. The APTT is indicated (by means of Pathromtin
 SL) in sec in various mixtures (3+1, 1+1, 1+3; sample+NPP) with a normal
 plasma pool (NPP).

Plasma sample 3 + 1 1 + 1 1 + 3
 FVIII-MP 56.9 48.9 46.5
 anti-FVIII-P 125.4 88.5 64.8
 LA 1 142.3 123.7 105.5
 LA 2 137.7 118.0 101.6
 NPP + 63 mg/l annexin 184.3 171.2 139.6
 NPP + 7.5 mg/l annexin 81.6 63.5 56.6
 In the case of a mixture of 1 part of normal plasma pool with 3 parts of
 the plasmas ("3+1" in Table 5), all pathological plasmas as well as the
 simulated lupus anticoagulant-positive plasma react pathologically. In the
 case of a 1+1 dilution, the deficiency in the factor-deficient plasma is
 already completely compensated (upper limit of the normal range 50 sec).
 The two natural lupus anticoagulant-positive plasmas, the
 annexin-containing plasmas, as well as the factor inhibitor-containing
 plasma are still highly pathological in the test even at higher dilution
 (1+3).