Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving blood clotting factor
Reexamination Certificate
2002-01-16
2004-05-04
Gitomer, Ralph (Department: 1651)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving blood clotting factor
C436S069000
Reexamination Certificate
active
06730490
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to in vitro methods, kits and reagents for the qualitative screening and quantitative determination of the functional activity of components of the Protein C anticoagulant pathway of blood coagulation.
BACKGROUND OF THE INVENTION
Maintenance of proper hemostasis is the result of a careful balance between procoagulant and anticoagulant activities. After a trauma, coagulation is triggered primarily through activation of coagulation Factors IX and X (FIX, FX) by tissue factor (also denoted tissue thromboplastin) and Factor VII (FVII) followed by generation of thrombin, which in turn cleaves fibrinogen to form soluble fibrin. After crosslinking by Factor XIII, a three-dimensional insoluble gel clot is obtained which prevents further blood losses.
Regulation of this highly potent system, shown schematically in
FIG. 1
, is accomplished by a balanced relation between procoagulant activities (shown with solid line arrows) and anticoagulant activities (shown with dashed line arrows). The anticoagulant activities include (1) inhibition of thrombin by antithrombin (AT) and &agr;
2
-macroglobulin, and (2) prevention of further thrombin formation by the Protein C anticoagulant pathway. In that pathway, activated Protein C (APC) inactivates the coagulation proteins Factor VIII and Factor V in their activated forms (FVIIIa, FVa) through proteolytic cleavage. In addition, Factor Xa is inhibited by antithrombin and tissue factor pathway inhibitor (TFPI), the latter also inhibiting the tissue factor/Factor VIIa complex. Factor VIIIa and Factor Va have potent procoagulant activities as cofactors in the activation of Factor X and prothrombin, respectively, and increase the reaction rates of these processes about 1000-fold each. Therefore, the inactivation of Factors Va and VIIIa by APC essentially stops further thrombin generation, thus providing a strong anticoagulant effect. Protein S and Factor V act as cofactors to activated Protein C (APC).
As shown in
FIG. 1
, the activation of coagulation through the intrinsic or extrinsic systems results in the activation of Factor X, a key component in the final common pathway. In the intrinsic system, the initial event is the activation of contact factors (Factor XII, prekallikrein) followed by the activation of Factor XI, which in turn activates Factor IX. In the extrinsic system, Factor IX and Factor X are activated by the tissue factor/Factor VIIa complex.
As
FIG. 1
shows, calcium ions have to be present in several of these reactions. The activation of Factor X by Factor IXa, and of prothrombin by Factor Xa, also requires procoagulant phospholipids. In vivo, this is provided by the membrane surface of activated platelets; in vitro by platelet extracts, purified phospholipids, synthetic phospholipids and/or crude phospholipid extracts from suitable sources. The total and free calcium ion concentrations in native plasma are about 2.4 and 1.2 mmol/L, respectively. Typically, calcium ion concentrations used in analytical methods for the determination of coagulation or anticoagulation factors are in the range 1.5-10 mmol/L. The concentrations of other metal ions in plasma are lower, with typical values for the total concentration being 1 mmol/L for Mg
2+
and 5-40 mmol/L for Zn
2+
, Cu
2+
and Mn
2+
.
Defects in the Protein C anticoagulant pathway may increase the risk of thrombosis due to a decreased capacity to prevent thrombin formation. Such defects may be due to deficiencies in the activity of Protein C and/or its cofactor Protein S. Another recently detected defect is a point mutation in the Factor V gene (G→A) at nucleotide 1691, resulting in the amino acid substitution Arg (R)→Gln (Q) at position 506 in Factor V/Factor Va, denoted FV:Q
506
or Factor V Leiden. Heterozygosity and homozygosity for this mutation are often denoted FV:R506Q and FV:Q506Q, respectively. This mutation is at one of the three APC cleavage sites (amino acids 306, 506, 679) in Factor Va, impairs its degradation by activated Protein C (APC), and confers a condition denoted as APC resistance.
APC resistance is to be considered a blood coagulation disorder recognized by an abnormally low anticoagulant response to activated Protein C (APC), and the determination of APC resistance may be used to screen for and diagnose thromboembolic diseases, such as hereditary thrombophilia, or for determining the risk for a human to acquire a manifestation of this blood coagulation disorder (e.g., European Pat. No. 608235).
Hence, there is a need to investigate these components of the Protein C anticoagulant pathway in the evaluation of thrombotic patients, and potentially also to screen for abnormalities of Protein C, Protein S and Factor V anticoagulant activity in situations connected with an increased risk of thrombosis, such as before surgery, during and after trauma, during pregnancy, or in connection with the use of oral contraceptive pills or hormone replacement therapy. Currently, clotting and/or chromogenic assays are available for analysis of Protein C and Protein S activity as well as for the detection of APC resistance (at least 90% of which is due to the FV:Q
506
mutation).
Protein C activity is typically measured after activation of endogenous Protein C, contained in a plasma sample, by thrombin or by a snake venom enzyme from
Agkistrodon contortrix contortrix
(e.g., European Patent 203509 to Stocker), commercially available as the reagent Protac®C (Pentapharm AG, Basel, Switzerland). The concentration of Protac®C in the activation mixture is typically about 0.1 U/mL or higher since otherwise an insufficient activation of Protein C may be obtained (Martinoli et al. (1986),
Thromb. Res.
43:253-264; McCall et al. (1987),
Thromb. Res.
45:681-685).
After activation by Protac®C, the protein C activity is determined with a clotting or chromogenic assay (Bertina (1990),
Res. Clin. Lab.
20:127-138; Marlar et al. (1989),
Hum. Pathol.
20:1040-1047; European Pat. No. 486515). In clotting methods, coagulation is triggered through the intrinsic pathway by using APTT reagents or through the extrinsic pathway with the use of tissue factor. In both cases calcium ions are added to a final concentration of usually 5-10 mmol/L. Commercial kits and reagents are available for the determination of Protein C activity, such as Acticlot™ C (American Diagnostica GmbH, Pfungstadt, Germany), Stachrom Protein C (Diagnostica Stago, Asnières, France), Staclot Protein C (Diagnostica Stago, Asnières, France), Coamatic® Protein C (Chromogenix AB, Mölndal, Sweden) and Protein C Activator (Dade Behring, Deerfield, Ill.).
The activation of Protein C by thrombin is stimulated about 1000-fold by thrombomodulin, an endothelial cell membrane protein (Esmon et al. (1981),
Proc. Natl. Acad. Sci
. (USA) 78:2249-2252). The use of thrombin/thrombomodulin as activator of Protein C for analysis of Protein C and/or Protein S activity in plasma samples, utilizing a photometric method, is also known (French Pat. Appin. No. 2689 640-A1).
Protein S activity is determined from its stimulation of the activity of APC in its degradation of Factor Va and/or Factor VIIIa. Typically, in such assays a standardized amount of APC is added to a plasma sample or activation of endogenous protein C is performed whereafter the clotting time is determined after a simultaneous or separate coagulation activation via the intrinsic system using an APTT reagent, via the extrinsic system using tissue factor or Factor Xa (Bertina (1990), supra; Preda et al. (1990),
Thromb. Res.
60:19-32; D'Angelo et al. (1995),
Thromb. Res.
77:375-378). Chromogenic activity assays for protein S have also been published, utilizing Factor IXa as an activator and monitoring Factor Xa generation (European Pat. No. 567 636) or thrombin generation (European Pat. No. 486 515). In all these methods, calcium ions are added as mentioned above.
The FV:Q
506
mutation in the Factor V molecule may be detected with molecular biology methods based upon the use of the polymerase
Hall Christina Maria Yvonne
Rosén Bert Steffen
Gitomer Ralph
Instrumentation Laboratory S.p.A.
Testa Hurwitz & Thibeault LLP
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