Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving blood clotting factor
Patent
1999-04-30
2000-07-11
Gitomer, Ralph
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving blood clotting factor
436 69, 530331, C12Q 156, G01N 3350, C07K 508
Patent
active
060871199
DESCRIPTION:
BRIEF SUMMARY
The invention concerns a method for determining the catalytic activity of factor IXa. The method according to the invention is suitable for finding factor IXa inhibitors (screening), for modulating blood coagulation (therapeutic application) and for determining factor IX and factor IXa in body fluids (diagnostic application).
Blood plasma proteases play a role in blood coagulation, wound closure by fibrin formation as well as in fibrinolysis i.e. clot lysis. After an injury the "injury signal" is amplified by the sequential activation (specific proteolysis) of inactive proenzymes to form active enzymes which initiates blood coagulation and ensures a rapid wound closure. Blood coagulation can be initiated by two paths, the intrinsic path in which all protein components are present in the blood and the extrinsic path in which a membrane protein, the so-called tissue factor plays a critical role.
The molecular mechanism of blood homeostasis (blood coagulation, fibrinolysis and the regulation of this equilibrium) and the components that are involved in this are comprehensively described in several review articles (Furie, B. and Furie, B. C., Cell 53 (1988) 505-518; Davie, E. W. et al., Biochem. 30 (1991) 10363-10379; Bergmeyer, H. U. (ed.): Methods of Enzymatic Analysis, Vol. V, chapter 3, 3rd ed., Academic Press, New York (1983)).
The factors of the blood coagulation cascade are very complex proteins. As a rule they can only be isolated in a complicated manner from the natural raw material source, the blood plasma, in a limited amount, with varying quality, homogeneity and purity (Van Dam-Mieras, M.C.E. et al., In: Bergmeyer, H. U. (ed.), Methods of Enzymatic Analysis, Vol. V, 3rd ed., page 365-394, Academic Press, New York (1983)). They play an important role in the regulation of blood homeostasis which is the equilibrium between blood coagulation, clot formation and dissolution. This well-regulated system can become unbalanced by genetic defects such as haemophilia A (defective factor VIII) and haemophilia B (defective factor IX). Acute disorders can lead to cardiac infarction, embolism and stroke.
There is therefore a need for substances which can influence the system of blood coagulation and fibrinolysis according to the medical requirements. For example from blood isolated or recombinantly produced factor VIII or factor IX are used to treat haemophilia A and B. tPA (tissue type plasminogen activator) and streptokinase (bacterial plasminogen activator) are used for example for clot lysis e.g. after cardiac infarction. In addition to complex proteins, substances such as hirudin (peptide composed of 65 amino acids, thrombin inhibitor), heparin (heteroglycan, cofactor of endogenous inhibitors) and vitamin K antagonists (inhibitors of .gamma.-carboxylation of Glu residues of the GlA domain) are also used to inhibit blood coagulation. However, the available substances are often still very expensive (protein factors) and not ideal with regard to their medical application (side effects) so that there is a need for medicaments which can be used to specifically modulate blood coagulation and clot lysis.
The search for new modulators (activators, inhibitors) of blood coagulation, fibrinolysis and homeostasis can for example be carried out by screening substance libraries and subsequently improving an identified lead structure by drug modelling. For this it is necessary that i) a suitable test and ii) the key protein(s) [target(s)] are available in an adequate amount and quality for screening and for crystal structure investigations (e.g. improvement of the lead structure by the specific prediction of changes based on the 3D structure of the protein component and lead structure).
Factor IXa (FIXa) is an interesting target for an inhibitor screening in order to find inhibitors to modulate blood coagulation. The known clinical picture of haemophilia B (factor IXa defect) warrants the assumption that specific factor IXa inhibitors are superior to known thrombin inhibitors with regard to the quite considerable pleiotrop
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Epstein William H.
Gitomer Ralph
Johnston George W.
Moran Marjorie A.
Parise John P.
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