Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving blood clotting factor
Reexamination Certificate
2000-01-19
2002-07-30
Gitomer, Ralph (Department: 1623)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving blood clotting factor
C435S023000, C435S975000
Reexamination Certificate
active
06426192
ABSTRACT:
The present invention relates to a method for the sensitive and functional detection of disorders in the protein C system (protein C, protein S, FV) and in particular for the determination of the activated blood coagulation factor V (FVa) with increased stability in respect of the decomposition by activated protein C (APC).
Hemostasis after vascular injuries results from an interaction between tissue, blood cells (blood platelets) and proteins of the blood liquid (plasma clotting factors, calcium ions). This interaction first leads to the formation of a hemostatic platelet plug (primary hemostasis) and finally to its consolidation by coagulation, i.e. by forming a network of insoluble fibrin. The physiological calcium content in blood of 60-70 mg per liter is essential for the optimal progress of the blood coagulation reactions. The fibrinolytic system is responsible for the enzymatic decomposition of fibrin clots during wound healing and recanalization of closed vessels. These interfering systems are modulated by activators and inhibitors and are present in the healthy organism in a labile balance. Disturbances of this balance may lead on the one hand to increased bleeding tendency and on the other hand to thrombosis proneness. Hemostatic disorders cause or accompany many diseases and therapies, in which cases the balance may be disturbed either in favour of bleeding or of thrombosis.
The section of the blood coagulation cascade represented in
FIG. 1
is of particular importance for the present invention. A detailed report on the reaction cascade leading to blood coagulation can be found in H. R. Roberts, Overview of the Coagulation Reactions, in: K. A. High and H. R. Roberts (eds.) Molecular Basis of Thrombosis and Hemostasis, pp. 35-50, Marcel Dekker: New York, Basel, Hong Kong (1995).
After injury of the vessel wall, blood comes into contact with tissue cells that release on their surface a glycoprotein of 50,000 Dalton which, as the so-called thromboplastin or tissue factor (TF), activates the blood clotting system via the exogenous pathway. Blood platelets adhering to tissue structures release phospholipids which activate the intrinsic blood coagulation system. TF forms with factor (F) VII present in plasma a complex that activates the zymogens FX and FIX to the serine proteases FXa and FIXa. Via the endogenous pathway, FIXa together with its cofactor FVIIIa also forms an efficient activator for FX in the presence of calcium ions and phospholipids. FXa forms with FVa, calcium ions and phospholipids a complex (prothrombinase complex) which converts the zymogen prothrombin into catalytically active thrombin. The enzyme thrombin converts fibrinogen by limited proteolysis into fibrin monomer which spontaneously polymerizes into fibrin.
The factors Va and VIIIa are non-enzymatic plasma proteins that strongly accelerate the activation of FX and prothrombin, respectively, by FIX and FXa, respectively. The catalytic efficacy of the cofactor, FVa, on the prothrombin activation is shown in Table 1. It can be seen that the activation of prothrombin by FXa, phospholipids and calcium ions is accelerated about 250 times alone by the presence of FVa.
The presence of calcium ions and phospholipids is a condition sine qua non for most reactions of the blood coagulation cascade. Removal of calcium ions by complexation, precipitation or ion exchange totally inhibits the clotting capacity of blood. This property is generally used to obtain blood plasma for analytical or therapeutic purposes by mixing freshly collected blood with sodium citrate for the complexation of calcium ions, centrifuging and decanting the supernatant, unclottable blood liquid from the sedimented cells. The coagulability of citrate plasma is restaured by addition of a physiological quantity of a calcium salt, by so-called recalcification.
TABLE 1
Catalytic efficacy of the prothrombinase complex
Prothrombin activation by the prothrombinase complex
Xa
1
Xa, Ca
2+
1.7
Xa, Ca
2+
, PL,
8.3 × 10
3
Xa, Ca
2+
, PL, Va
2.0 × 10
6
The anticoagulant protein C system prevents an uncontrolled migration of activated blood clotting factors from the site of the vascular injury and hemostasis. As in the plasmatic coagulation system, a cell-bound receptor, thrombomodulin (TM), and a non-enzymatic cofactor, protein S, as well as the secondary components phospholipids and calcium ions participate in the activation of the protein C system. Thrombin escaping from the site of hemostasis binds to TM, loses thereby its fibrinogen-coagulant properties and becomes the specific activator for the zymogen protein C. Activated protein C (APC) is a serine proteinase which, potentiated by protein S, splits off and inactivates the clotting factors FVa and FVIIIa. By inactivation of these cofactors, the coagulation process is strongly slowed down: according to Table 1, a 250-fold delay takes place alone by the inactivation of FVa. For a topical description of the protein C system, see K. Suzuki, Protein C: in: K. A. High and H. R. Roberts (eds.) Molecular Basis of Thrombosis and Hemostasis, pp. 393-424, Marcel Dekker: New York, Basel, Hong Kong (1995).
The biological significance of the protein C system has been evidenced in 1993 by B. Dahlback who observed that in patients with thrombosis tendency, unlike in healthy subjects, the activated partial thromboplastin time (APTT, a diagnostic function control for the endogenous coagulation system) is not prolonged after addition of activated protein C. He defined his observations as “resistance against activated protein C” (APC resistance). This resistance is in 97% of the cases due to a point mutation in the factor V gene. This genetically inherited defect can be found in about 5% of the normal population and in at least 20% of young patients with first unexplainable or recurrent thromboembolisms. In the presence of the mutation, the activated clotting factor V can no more be split and thus inactivated. Consequences of the deficiency of this particularly important anticoagulant component of the blood coagulation system may be coronary heart diseases, venous thromboses or thromboembolisms. Consequently, heterozygous defect carriers present a 5-10 times greater thrombosis risk than normal persons and homozygous defect carriers an even 50 to 100 times higher risk.
Other hereditary or acquired deficiencies or defects in the protein C system (qualitative or quantitative protein C or protein S deficiency) are also associated with higher thrombosis tendency.
The congenital or acquired APC resistance can be detected by a functional test or by the direct detection of the mutation on the DNA level (genotype).
The functional detection can be performed according to Dahlback by an APTT variant (PCT/SE92/00310; WO 93710261) in which the coagulation of a platelet-free plasma sample is once triggered off once by calcium chloride without addition of activated protein C (APC) and once by calcium chloride with addition of APC. The thrombin quantity resulting in the test mixture is determined either by the conversion of the natural substrate fibrinogen into a clot (clotting time) or photometrically by the release of a chromophore from a chromogenic substrate. The existence of the factor V mutation can be noted by the fact that the clotting time is only weakly prolonged by APC, while APC strongly prolongs the APTT of normal plasma. Dividing the clotting time of the sample with APC by the clotting time of the sample without APC gives a ratio of diagnostic significance. A ratio of more than 2.0 is found in healthy subjects, a ratio between 1.3 and 2.0 in heterozygous and a ratio below 1.3 in homozygous defect carriers. However, as this test system bases on the activation of coagulation in the presence of calcium ions, quantitative and qualitative abnormalities on calcium-dependent plasma Table 1. Catalytic efficacy of the prothrombinase complex clotting factors (FII, VII, VIII, IX, X) can falsify the result. Deficiency or dysfunction of protein S can give erroneously positive values and
Gempeler-Messina Patrizia
Müller Christian
Stocker Kurt
Chaudhry Mahreen
Gitomer Ralph
Pennie & Edmonds LLP
Pentapharm AG
LandOfFree
Method for the functional detection of disorders in the... 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 functional detection of disorders in the..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Method for the functional detection of disorders in the... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2908227