Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving blood clotting factor
Reexamination Certificate
2001-11-21
2004-05-25
Nashed, Nashaat T. (Department: 1652)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving blood clotting factor
C435S018000, C435S023000, C435S024000
Reexamination Certificate
active
06740496
ABSTRACT:
FIELD OF THE INVENTION
The present invention is in the field of diagnostics and relates more in particular to a method for the determination of biologically active forms of proteolytic enzymes, such as thrombin, in blood and other fluids.
BACKGROUND OF THE INVENTION
In the Western world, arterial and venous thrombosis and atherosclerosis together are currently responsible for well over 50% of all mortality and serious morbidity. In a few decades this will be the case world-wide [Murray, C. J. and A. D. Lopez, Science (1996) 274:740-743]. Thrombosis is caused by an overactivity of the haemostatic mechanism that is responsible for the arrest of bleeding from a wound. This haemostatic-thrombotic system (HTS) is a complex interaction between vessel wall, blood cells, especially blood platelets, and plasma proteins. See, e.g., Hemker H. C. Thrombin generation, an essential step in haemostasis and thrombosis. In: Haemostasis and thrombosis, A. L. Bloom et al., eds., Churchill Livingstone, Edinburgh, (1994) 477-490.
In blood there are at least three series of proenzyme-enzyme cascades of great biological importance: the blood coagulation system, the fibrinolytic system and the complement system. In each of these systems proteolytic proenzymes are activated and subsequently inhibited. In general it is important that the concentration of an activated enzyme in such a cascade can be measured in order to assess the function of the system. In the following a typical description of the clotting system will be given with an emphasis on its most important enzyme, thrombin. It is to be noted, however, that the present invention also relates to other clotting enzymes and enzymes of the fibrinolytic and complement pathway.
The mechanism of thrombin generation can be outlined in some detail as follows. In the plasmatic coagulation system factor Xa is formed by the action of the tissue factor-factor VIIa complex (TF-VIIe). Factor Xa binds to tissue factor pathway inhibitor (TFPI) and the TFPI-Xa complex inhibits the TF-VIIa complex. Thrombin activates factors V, VIII and XI and so accelerates its own generation, but it also binds to thrombomodulin and so starts the protein C mechanism that breaks down factors V and VIII, thus, indirectly, inhibiting further thrombin generation. An important fraction (≈30%) of all thrombin formed in clotting plasma is bound to the fibrin clot. Clot-bound thrombin does retain its thrombotic properties, it can clot fibrinogen, activate factors V, VIII and XI as well as platelets [Béguin, S. and R. Kumar, Thromb. Haemost. (1997) 78:590-594; Kumar, R., S. Béguin, and H. C. Hernker, Thromb. Haemost. (1994) 72:713-721, and (1995) 74:962-968]. It is not inhibited by antithrombin.
Thrombin action causes receptors in the platelet membrane to bind fibrinogen, which causes platelet aggregation. Platelet activation also leads to the exposure of procoagulant phospholipids in a Von Willebrand factor dependent ii reaction [Béguin S., R. Kumar, I. Keularts, U. Seligsohn, B. C. Coller and H. C. Hemker, Fibrin-Dependent Platelet Procoagulant Activity Requires GPlb Receptors and Von Willebrand Factor, Blood (1999) 93:564-570; Béguin, S. and R. Kumar, supra (1997)]. These phospholipids are required for the proper activation of factor X and prothrombin. Recently, the picture has been complicated by the discovery that fibrin, previously thought to be the inert endproduct of coagulation, plays an active role itself. It binds and activates Von Willebrand factor, which activates platelets and provokes the exposure of procoagulant phospholipids via an alternative pathway.
The cooperation between platelets and the coagulation system, including fibrin, is central to the haemostatic-thrombotic system. The mechanism shows an abundance of positive and negative, often nested, feedback loops. Underactivity causes bleeding, overactivity causes thrombosis. Thrombosis manifests itself as coronary infarction, stroke, pulmonary embolism and a large number of less frequent diseases.
In order to assess the function of such a system, also for diagnostic purposes and for the safe use of antithrombotic drugs, a probe is needed for the functional status of the haemostatic-thrombotic system. An important function test of the HTS is the thrombin generation curve (TGC). Carried out in platelet-poor plasma, it gives information about the function of the plasmatic clotting system. In platelet-rich plasma it measures also the function of the platelets.
According to the prior art the TGC can be measured by subsampling or continuously. In the ancient subsampling method [Biggs, R. and R. G. Macfarlane,
Human Blood Coagulation and its Disorders
. 1953, Oxford: Blackwell; Quick, A. J.,
Haemonhagic Diseases
. 1957, Philadelphia: Lea & Febiger], samples are taken from a clotting mixture, and the concentration of thrombin is measured in each sample.
Active thrombin survives in plasma for only a limited period of time (the half life time is 16-17 s). This is due to circulating antithrombins. Most thrombin (64%) is inactivated by antithrombin (AT), a plasma-protein of 57 kD, 23% by &agr;
2
-macroglobulin (&agr;
2
M), a 725 kD plasma-protein, and 13% by various other agents [Hemker, H. C., G. M. Willems, and S. Béguin, A computer assisted method to obtain the prothrombin activation velocity in whole plasma independent of thrombin decay processes, Thromb. Haemost. (1986) 56:9-17]. The &agr;
2
-macroglobulin-thrombin complex (&agr;
2
M-IIa) has the peculiarity that it is inactive towards all macromolecular substrates, but retains its activity against small molecular weight (artificial) substrates. &agr;
2
-Macroglobulin is a glycoprotein of Mr 725,000, which is present in plasma in a concentration of 2500 mg/L or 3.5 &mgr;M. It is a tetramer of identical subunits of 185 kD. The inactivation reaction of an active proteinase or activated clotting factor with &agr;
2
M is a three step process: 1°. Formation of a loose complex, 2°. Hydrolysis of a target peptide in &agr;
2
M, causing 3°, a rapid conformational change which physically entraps the enzyme molecule within the &agr;
2
M molecule. [See for a review: Travis, J. and G. S. Salvesen, Human plasma proteinase inhibitors. Ann. Rev. Biochem. (1983) 52:655-709].
The group of Hemker then developed a continuous method in which thrombin-catalysed product formation from suitable substrates is monitored directly [Hemker, H. C., et al., Continuous registration of thrombin generation in plasma, its use for the determination of the thrombin potential. Thromb. Haemost. (1993) 70:617-624]. The kinetic constants of the substrate are such that, at the concentration used, the rate of product formation is proportional to the amount of enzyme present (thrombin or &agr;
2
M-IIa). The time-course of enzyme activity in the sample can be estimated as the first derivative of the product-time curve.
A drawback of this method is that part of the thrombin in plasma binds to &agr;
2
-macroglobulin. &agr;
2
-Macroglobulin is the most abundant non-specific protease inhibitor of blood plasma. It quenches the biological activity of proteases (activated clotting factors, activated fibrinolytic enzymes and activated complement factors) without occupying their active centre, so that there is a residual activity on the usual oligopeptide signal-substrates.
This &agr;
2
-macroglobulin-thrombin complex has no known physiological activity but is able to cleave chromogenic substrates. Thus, the product formation observed is the result of the combined activities of free thrombin and &agr;
2
-macroglobulin-bound thrombin. The relevant data, i.e., the amount of product formed by free thrombin only, can be extracted from the experimental product formation by a mathematical operation. However, the operation has to be carried out on the whole course of the product generation curve. The product formation therefore needs to be monitored continuously. Although the principle of this continuous method is applicable to all substrates that give a prod
Hemker Hendrik C.
Ramjee Manoj
Wagenvoord Robert J.
Kalow & Springut LLP
Nashed Nashaat T.
Synapse B.V.
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