Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving blood clotting factor
Reexamination Certificate
1998-09-30
2003-05-06
Whisenant, Ethan (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving blood clotting factor
C435S006120, C536S023100, C536S024300
Reexamination Certificate
active
06558913
ABSTRACT:
The subject invention lies in the field of haemostasis and in particular is directed at the aspect of thrombosis. More particularly the invention is directed at a method for screening and diagnosis of thrombophilia, especially hereditary thrombophilia. The method according to the invention can then be used for determining the risk for thrombosis in individuals.
BACKGROUND TO THE INVENTION
Deep vein thrombosis is a common disease. Well established risk factors include recent surgery, malignant disorders, pregnancy and labour, long term immobilisation, and deficiency of one of the main inhibitors of the clotting system (Ref. 1). The main inhibitors are known to be protein C, protein S and antithrombin. The causes of deep vein thrombosis in many patients remain unclear. It has recently been established however that a poor anticoagulant response to activated protein C (APC) is present in several families with a hereditary tendency to venous thrombosis (Ref. 2).
The anticoagulant property of APC resides in its capacity to inactivate the activated cofactors Va and VIIIa by limited proteolysis (Ref. 3). This inactivation of cofactors Va and VIIIa results in reduction of the rate of formation of thrombin, the key enzyme of coagulation. In vitro, this effect can be visualised by adding APC to normal plasma and accordingly determining the effect thereof in a coagulation test, for example in a test determining the APTT (activated partial thromboplastin time). Activation of protein C occurs at the surface of endothelial cells via the thrombin-thrombomodulin complex (Ref. 27). Thrombomodulin is a membrane glycoprotein that can bind thrombin. By this binding thrombin loses the ability to convert fibrinogen to fibrin and the ability to activate blood platelets. In other words thrombin loses its coagulant properties and reduces its further own production (so-called negative feed-back) by activating protein C. In vivo (in the presence of calcium) the activation of protein C is almost completely dependent on the availability of thrombomodulin on the endothelium. APC is subsequently neutralized by formation of complexes with APC inhibitor (PCI) and &agr;
1
antitrypsin, which means that in normal conditions it remains only for a short time in the circulation and the anticoagulant effect remains generally locally expressed.
It was generally accepted that the inactivation of the cofactors Va and VIIIa by APC proceeds only optimally in the presence of Ca
2+
, phospholipids and the APC cofactor protein S (Ref. 4, 28, 29). More recently this view was, however, challenged by the finding that in systems of purified proteins protein S has little cofactor activity to APC (Ref. 5, Ref. 6). A possible solution for this apparent discrepancy between the observations in vivo (thrombotic tendency in hereditary protein S deficiency) and in vitro (poor APC cofactor activity of protein S in systems of purified proteins) could be offered by the finding of Dahlbäck et al (Ref. 2) who reported patients with normal values for antithrombin activity, protein C (immunologically and functionally) and protein S (immunologically and functionally) without indications for abnormal plasminogen, abnormal fibrinogen or lupus anticoagulants, but with a reduced anticoagulant response to activated protein C. The latter was found with a new test developed by Dahlback (Ref. 2) in which he studies the response (coagulation time, APTT) of a plasma after in vitro addition of purified human APC. The addition of activated protein C to the plasma of these thrombotic patients did not result in the expected prolongation of the activated partial thromboplastin time (APTT). After postulating a number of mechanisms for this phenomenon only one was considered to provoke the poor anticoagulant response to APC, namely the existence of a hitherto unknown cofactor to APC that is deficient in these patients.
The following mechanisms have to date been rejected as being causes of the poor anticoagulant response to APC:
1. The presence of an auto antibody against APC
2. A fast acting protease inhibitor reacting with APC
3. A functional protein S deficiency
4. Mutations in the Factor V or Factor VIII gene
Dahlbäck (Ref. 2, 7) postulated that in the families studied a hereditary shortage of a hereto unknown APC cofactor that purportedly works independently of protein S was the cause of APC resistance. Dahlbäck et al (Ref. 2) also described a test method for diagnosing the thrombo embolic disorders by addition of activated protein C to a patient sample containing coagulation factors followed by measurement of an enzyme activity that is influenced by the addition of APC in an international patent application WO93/10261. It is stated in the application of Dahlbäck et al that the experimental results presented indicated that the disorders in question are related to a hitherto unknown coagulation factor or factors or unknown interactions of known factors. The unknown factor is not Factor Va or VIIIa that is resistant to degradation by APC and neither is it an inhibitor of the immunoglobulin type of APC. Furthermore it is not related to protein S deficiency. Dahlbäck et al (Ref. 2) state that their invention is a method particularly useful for further diagnosis of thromboembolic diseases such as hereditary or non hereditary thrombophilia and for determining a risk for thrombosis in connection with pregnancy, taking anticonception pills, surgery etc. They describe their method as being characterized in that the coagulation system in a sample is activated, wholly or partly in a manner known per se and incubated with activated protein C, whereupon a substrate conversion reaction rate like clotting or conversion of a chromogenic substrate is determined. The conversion rate obtained is compared with values obtained for normal plasma samples. If the rate is enhanced it indicates that the individual from which the sample is derived may suffer from a clotting disease. The disease is not expressed by protein S deficiency or by formation of Factor Va or Factor VIIIa resistant to degradation by APC or by an inhibitor of the immunoglobulin type for APC. In the international application it is also stated by Dahlbäck et al that the data in the application indicated that the patient in question could not carry a defective Factor VIII/VIIIa in contrast to what they had previously stated in Thromb. Haemostas. 65, Abstract 39, 658 (1991), wherein addition of activated protein C to a plasma sample of a patient, and study of the effect produced was claimed to have illustrated a defective Factor VIIIa molecule not degraded by activated protein C. Furthermore in the international patent application the assay was used to directly measure the inhibition of Factors Va and VIIIa by APC. Using the Factor Xa based clotting assay described therein, the inhibition of patient Factor Va by APC was found to be normal suggesting that Factor Va in the patient's plasma was degraded in a normal fashion by exogeneously added APC.
Following the publication by Dahlbäck et al (Ref. 2) other groups started research in this area. In Blood Vol. 82, nr. 7, 1993 on pages 1989-1993 Griffin et al describe the results of APC resistance tests carried out among 25 venous thrombotic patients with no identifiable blood coagulation abnormality and 22 patients previously identified with heterozygous protein C or protein S deficiency. The APC induced prolongation of the activated partial thromboplastin time assay for these patients was compared with results for 35 normal subjects. The results showed that his new defect in antiocoagulant response to APC was surprisingly present in 52 to 64% of the 25 patients i.e. in the majority of previously undiagnosed thrombophilia cases. The deficiency was not present in 20 of 22 heterozygous protein C or protein S deficient patients. This suggested that the new factor is a risk factor independent of protein C or protein S deficiency. Mixing of normal blood plasma with each of two extremely defective plasmas (APC-induced prolongation of APTT<20 s) was performed an
Bertina Rogier Maria
Reitsma Pieter Hendrik
Akzo Nobel N.V.
Whisenant Ethan
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