Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving blood clotting factor
Patent
1994-05-27
1995-08-22
Beisner, William H.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving blood clotting factor
435810, 435975, 436 69, C12Q 156, G01N 3386
Patent
active
054439604
DESCRIPTION:
BRIEF SUMMARY
The present invention concerns a novel method appropriate for screening and diagnosis of thromboembolic diseases, e.g. hereditary thrombophilia. The invention can also be used for determining the risk for thrombosis in pregnant individuals, individuals undergoing surgery, individuals taking anti-conception drugs etc.
The blood coagulation comprises a complex system of inter-linked proenzymes, enzymes and cofactors performing its role at the surfaces of activated platelets and endothelial cells. When the system is activated the ultimate result is the formation of a blood clot containing insoluble fibrin. The initiation and termination of fibrin formation is carefully regulated in normal homeostasis. In vitro, platelets and endothelial cell surfaces are usually substituted with suitable phospholipids. For the invention the relevant part of the coagulation system is described in PCT WO93/10261.
The important part for the invention relates to Protein C (=PC) and effects produced by action of activated Protein C (=APC) on the coagulation system. Protein C is a zymogen that in vitro may be activated by thrombin (=Factor II.sub.a) alone, the combination thrombin-thrombomodulin, certain snake venoms, such as from Akistrodon Contortrix Contortrix, or purified Factor X.sub.a. Activation of sample endogenous Protein C or addition of exogenously activated Protein C to a plasma sample will neutralise Factors V.sub.a and VIII.sub.a (degradation) and lead to prolonged times for blood coagulation in plasma samples of healthy individuals. Factors V and VIII are activated by small amounts of thrombin or Factor X.sub.a. Protein S is a cofactor to Protein C.
Hereditary heterozygous deficiences in Protein C, Protein S and Antithrombin III (ATIII, a coagulation factor opposing coagulation) are found in approximately 10-15% of patients with diagnosed thromboembolic disease before the age of 40. Homozygous Protein C and S deficiencies are life threatening and affected individuals develop generalized microvascular thrombosis and purpura fulminance in the neonatal period.
Deficiencies in Protein C and S and ATIII are measured by both functional and immunological methods. One way of determining functional Protein C activity involves the steps: mixing the plasma sample to be tested with an excess of human plasma deficient in Protein C and addition of a Protein C activator and monitoring the appropriate substrate conversion. Highly specific assays have been achieved by using Protein C specific substrates. Alternatively one has also utilized substrates for enzymes, e.g. thrombin and Factor X.sub.a, which activities are influenced by activated Protein C activity (i.e. enzyme activities that are generated or modulated by APC). In certain cases Protein C assays have involved isolation of the zymogen and a subsequent activation and addition of substrate to the activated form. Measurement of Protein C activity in plasma samples has also been suggested to be performed directly in the sample without addition of plasma deficient in Protein C. However, such methods will not discriminate an abnormality related to Protein C as such from a disorder related to factors interfering with the effects caused by Protein C (WO-A-91/02812).
Addition of activated Protein C to a plasma sample of a patient and study of the effect produced has been claimed to discover a defect Factor VIII.sub.a molecule that is not degraded by activated Protein C (B. Dahlback and M Carlsson, Thromb. Haemost. 65, Abstract 39, 658 (1991)). However, the data given in the present specification surprisingly indicate that the patient in question could not carry a defect Factor VIII/VIII.sub.a.
In order to determine Protein S functional activity in a plasma sample the most common methods involve mixing the plasma sample with activated Protein C, an excess of Protein S deficient plasma and further reagents necessary to achieve clotting (Waart et al., Thromb. Res. 48, 427-37 (1987); Suzuki et al., Thromb. Res. 49, 241-51 (1988); Bertina et al., Thromb. Haemost. 53,268-72 (1985); and Co
REFERENCES:
patent: 5001069 (1991-03-01), Bartl
patent: 5051357 (1991-09-01), Hassouna
patent: 5059525 (1991-10-01), Bartl
patent: 5169786 (1992-12-01), Carroll
patent: 5308756 (1994-05-01), van de Waart et al.
Amer, L. et al., Impairment of the Protein C Anticoagulant Pathway in a Patient with Systemic Lupus Erythematosus, Anticardiolipin Antibodies and Thrombosis; Thrombosis Research 57; pp. 247-258, 1990.
Griffin, J. et al., Anticoagulant Protein C Pathway Defective in Majority of Thrombophilic Patients; Blood, vol. 82, No. 7 pp. 1989-1993, Oct. 1, 1993.
Thrombophilia: a new factor emerges from the mists; The Lancet, vol. 342, pp. 1501-1506, Dec. 18/25, 1993.
Faioni, E. M. et al., Resistance to Activated Protein C in Nine Thrombophilic Families: Interference in a Protein S Functional Assay; Thrombosis and Haemostasis, F. K. Schattauer Verlagsgesellschaft mbH (Stuttgart) 70 (6) 1067-1071 (1993).
Dahlback, B. et al., Inherited resistance to activated protein C is corrected by anticoagulant cofactor activity found to be a property of factor V; Proc. Natl. Acad. Sci. USA, vol. 91, pp. 1396-1400, Feb. 1994.
Bauer, Kenneth A., M.D., Hypercoagulability-A New Cofactor in the Protein C Anticoagulant Pathway; New England Journal of Medicine, vol. 330, pp. 566-567, Feb. 24, 1994.
Chromogenix, Coatest APC Resistance Kit Instructions May 1993.
Chromogenix, Coatest APC Resistance Kit Brochure Jun. 1993.
Chromogenix, Thrombophilia 1993-1994, Coatest APC-Resistance Brochure Oct. 1993.
Dahlback, B. et al.,Familial thrombophilia due to a previously unrecognized mechanism characterized by poor anticoagulant response to activated protein C: Prediction of a cofactor to activated protein C; Proc. Natl. Acad. Sci. USA, vol. 90, pp. 1004-1008, Feb. 1993.
Mitchell, C. et al., A Fatal Thrombotic Disorder Associated with an Acquired Inhibitor of Protein C; New England Journal of Medicine, vol. 317, No. 26, pp. 1638-1642, Dec. 24, 1987.
Thromb. Haemost. 65, Abstract 39, 658, 1991.
Takahashi H., Fast Functional Assay of Protein C . . . Clinica Chimica Acta 175 (1988) 217-226.
Vasse M., Protein C: Roen, A New Hereditary . . . Thrombosis Research 56: 387-398 1989.
Dahlback B., Factor VIII Defect Associated with . . . Thrombosis & Haemostasis 65 Abst 39 p. 658 Jul. 1991.
Beisner William H.
Gitomer Ralph
LandOfFree
Method for the diagnosis of blood coagulation disorders does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Method for the diagnosis of blood coagulation disorders, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Method for the diagnosis of blood coagulation disorders will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2141186