Chemistry: analytical and immunological testing – Clotting or clotting factor level tests
Patent
1997-04-24
2000-04-18
Soderquist, Arlen
Chemistry: analytical and immunological testing
Clotting or clotting factor level tests
436 63, 436 86, 436164, 436172, 436174, G01N 3386
Patent
active
060514345
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
The present invention relates to an improved test for protein C resistance in the blood coagulation system of patients.
BACKGROUND OF THE INVENTION
Known mechanisms for blood coagulation thrombosis and haemostasis are well described in International Patent Publication WO91/01382 the contents of which are incorporated herein by reference.
It is known from International Patent Publication WO93/01261, Bertina, R. M., Keulemans, B. P. C., Koster, T., et al, "Mutation in blood coagulation factor V associated with resistance to activated protein C", Nature 369, pp. 64 to 67 (1994) and Dahlback et al, "Inherited Thrombophilia: Resistance to activated protein C as a pathogenic factor of venous thromboembolism", Blood 85, pp. 607-614 (1995) that the risk of thrombosis in patients with a mutant factor V molecule known as the Leiden variant, or with activated protein C impairment for some other reason, may be determined by activating the coagulation system in a plasma sample and incubating the sample with activated protein C in what has come to be known as an activated protein C impairment, impedance or resistance test. There are precedents for this test in which impairment of activated protein C has been detected in patients with acquired thrombophilia (Mitchell et al, "Fatal thrombotic disorder associated with an acquired inhibitor of protein C", New England J. Med. 317, pp. 1638 to 1616 (1987) and Amer et al, "Impairment of the protein C anticoagulant pathway in a patient with systemic lupus erythematosus, anticardiolipin antibodies and thrombosis", Thromb. Res. 57, pp. 247-1990 (1988).
A substrate conversion reaction rate may be determined by the clotting time or by the time required for the conversion of a chromogenic substrate to a colored product. The conversion rate obtained is compared with values obtained in the absence of activated protein C and also with results for normal plasma samples. If the clotting time is not sufficiently prolonged by activated protein C, it indicates that the individual from which the sample is derived may be at a higher-than-normal risk of thrombosis.
SUMMARY OF THE INVENTION
According to the present invention if factor V is specifically activated by an exogenous reagent in addition to activation of the common pathway through factor X the test for activated protein C resistance may be made more sensitive and specific than previously known tests. According to the present invention a better specificity is obtained when a complex factor X activator is used together with the factor V activator. A similar result is achieved if prothrombin is activated to thrombin by a factor v dependent activator in the presence of a factor V activator.
The present invention consists in a method for determining the functional activity of protein C in a human plasma sample, comprising the steps of common pathway of the blood coagulation mechanism through factor X or by inducing the presence of thrombin in a factor V dependent manner, for efficient clotting of the plasma samples; of the plasma sample; and the equivalent rate determined for a normal patient, or comparing the potential rate of coagulation measured in step (b) with the equivalent rate determined for the plasma sample in the absence of activated exogenous protein C, and determining the functional activity of the free protein C from one or the other of those comparisons.
In a preferred form of the invention the patient's plasma sample is pre-incubated with an exogenous activator for factor V prior to the initiation of clotting. The exogenous activators for both factors X and V are most preferably derived from snake venom. In one embodiment of the invention both the factor V and the factor X activators are derived from Russell's viper. The factor X activator is preferably derived from the venom of Russell's viper (Vipera russelli) and other immunologically cross-reactive species. A preferred factor V activator derives from Naja nivea and other immunologically cross-reactive species. The snake venoms may either be
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Gradipore Limited
Soderquist Arlen
Striker Michael J.
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