Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving blood clotting factor
Reexamination Certificate
1999-02-05
2001-06-26
Gitomer, Ralph (Department: 1623)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving blood clotting factor
Reexamination Certificate
active
06251619
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a new use of a particular snake venom, the venom of
Crotalus viridis helleri
(this venom is referred to hereinafter as CVH), in the field of determining the reactivity of the activated protein C, this determination including that of the activity of the functional protein C and that of resistance to the activated protein C (activated protein C is called APC herein and resistance to APC is abbreviated as APC-R; the other abbreviations used are defined further below). The invention likewise relates to a method for determining APC-R, using CVH and a dosing kit enabling this method to be used.
PRIOR ART
The study of hereditary thromobophiliacs has shown that APC-R is mainly associated with a mutation on the gene coding for factor V. This mutation, called “Leiden's mutation,” “mutation R506Q” or “mutation
506
R→
506
Q,” appears in the sequence of the amino acids of human factor V by the replacement of Arg by Gln in position 506, and favors the appearance of thromboses, often venous (in the lower body members with, in some cases, pulmonary embolism). See particularly in this connection the publications of R. M. Bertina et al.,
Nature,
1994, 369, pages 64-68, of M. Kalafatis et al.,
J. Biol. Chem.
1994, 269, pages 31869-31880, of H. De Ronde et al.,
Thromb. Haemost.,
1994, 72, pages 880-886, of P. J. Svensson et al.,
N. Engl. J. Med.,
1994, 330, pages 517-522, of B. Dahlbäck et al.,
Proc. Natl. Acad. Sci. USA,
1993, 90, pages 1004-1008, and of B. Dahlbäck et al.,
Proc. Natl. Acad. Sci. USA,
1994, 91, pages 1396-1400.
Other factor V anomalies can be at the origin of APC-R. In particular, it is possible that an untimely cleavage in position 306 (i.e.,
306
Arg) of the amino acid sequence of factor V and/or Va is one of the other possible causes of APC-R.
The determination of APC-R was employed initially (particularly by an APTT, KCCT or other such test) to detect the existence of a thrombotic disorder by comparing the coagulation time, which is prolonged by the addition of APC, of a control plasma with one that is shorter in case of an irregularity in relation to the preceding one, of a sample of human plasma to be tested with APC added.
See in this connection: C. A. Mitchell et al.,
N. Engl. J. Med.,
1987, 317, pages 1638-1642, L. Amer et al.,
Thromb. Res.,
1990, 57, pages 247-258, and B. Dahlbäck et al.,
Thromb. Haemost.,
1991, 65, Abstract 39, page 658.
The only kit presently available on the market (it is sold by the name of “Coatest APC-Resistance” by the firm of Chromogenix) uses the process described in the disclosure of B. Dahlbäck et al.,
Thromb. Haemost.,
1991, 65, Abstract 39, page 658, referred to above and explained in WO-A-9310261, and the article by B. Dahlbäck et al.,
Proc. Natl. Acad. Sci. USA,
1993, 90, pages 1004-1008, referred to above. This process comprises mixing one volume of the plasma to be tested or of the control plasma with one volume of the APTT reactant [PL +surface activator (silica, kaolin or glass)], incubation for 4 minutes at 37° C., then starting coagulation by means of one volume of a solution of CaCl
2
, on the one hand, or of one volume of a solution of CaCl
2
and APC, on the other.
This process is unsatisfactory in that, being sensitive particularly to the presence of factor VIII, heparin, PS, CAC's and VKA's, it gives too many false negative and false positive results. Improvements have therefore been proposed in WO-A-9615457, WO-A-9604560 and EP-A-0711838.
WO-A-9615457 proposes an APC-R determination in which the plasma to be tested is placed in contact with a procoagulant reactant (tissue factor in this case) and a VdfP; then, after incubation, coagulation is started with CaCl
2
or CaCl
2
+APC, the variation of the coagulation time being then determined by comparison with a control plasma.
WO-A-9604560 aims at an improved technique for detecting APC-R. This technique is based on the use of an exogenic reactant that specifically activates the V factor to Va, to supplement an activation of the coagulation either by the transformation X→Xa, or by the transformation of prothrombin to thrombin by a mechanism depending on the V factor.
WO-A-9604560 recommends particularly,
as an exogenic reactant, a snake venom such as the venom of Naja nivea to activate V to Va, and
for the supplemental activation: either RVV-X to activate X to Xa, either a snake venom (or venom extract), such as the venoms of
Pseudonaja textilis, Notechis scutatus
or
Oxyurnus scutellatus,
for the activation of prothrombin to thrombin by a mechanism depending on factor V.
For the determination of APC-R, EP-A-0711838 recommends a process comprising;
(a) the mixing of a plasma to be tested with a reactant A with a low content of factor V, particularly VdfP,
(b) the addition of a reactant B that directly or indirectly activates factor V to Va, particularly a viper venom such as RVV (with by its RVV-V fraction induces the activation of V to Va and which by its RVV-X fraction induces the activation of X to Xa) or the venom of
Echis carinatus
(which induces the activation of prothrombin to thrombin and then activates V to Va indirectly).
c) the addition of a reactant C permitting degradation of the Va factor, viz., APC [(or the mixture PC +activator (particularly ACC)], and
d) the addition of reactants permitting the determination of the residual activity of the Va factor, on the condition that the proportion of the volume of the plasma sample to be tested with respect to the total volume is no more than 20%, advantageously less than or equal to 10% (“the content of the sample volume being a maximum of 20%, but preferably less than or equal to 10%,” according to claim
1
, page 13, lines 39-40 of EP-A-0711838).
This process has the disadvantage of calling for volumes of plasma (to be tested, or plasma for control), of reactant A and of reactant B which are all three different. This circumstance does not make it easy to measure the coagulation time with a fully automatic apparatus.
The aforesaid processes of WO-A-9604560 and EP-A-0711838 make it possible to relieve most of the disadvantages of the “Coatest APC-Resistance.” However, these two documents neither describe nor suggest the use of the particular snake venom of the invention, viz. CVH, to initiate coagulation at the level of the X factor by the transformation X→Xa.
The only effective technique today for determining APC-R is based on molecular biology (MB). It is lengthy, fussy, burdensome and calls for prepared material and highly qualified personnel. Moreover, its employment must satisfy Article L. 145-15 of French Law No. 94-653 of Jul. 29, 1994 which requires obtaining the written consent of the persons for whom a genetic study is undertaken.
THE SUMMARY OF THE INVENTION
It is intended to provide a new technical solution for the determination of APC-R which (i) will be simple at the level of practice, (ii) will be at least as effective as those considered in the above-mentioned documents WO-A-9604560 and EP-A-0711838, and (iii) is based on initiating coagulation at the X factor (i.e., transformation X-Xa) whose importance has been recognized in WO-A-9604560 (see page 5, lines 1-12).
The aim is to improve sensitivity by reducing the number of false positive and false negative results. With this in mind it is proposed to employ an APC-R determination process by classical mechanisms, but with the peculiarity that a new reactant or means is used to initiate coagulation by the transformation X-Xa, this means or reactant improving sensitivity.
The new technical solution according to the invention is based on the selection of a particular activator to initiate coagulation at the X factor (i.e., X→Xa). This particular activator is a snake venom, namely CVH.
According to a first aspect of the invention, a new use for a snake venom is announced, for starting coagulation by activating the X factor to Xa for the purpose of determining the reactivity of the activated protein C, part
Evenson, McKeown, Edwards & Lenahan P.L.L.C.
Gitomer Ralph
Societe Diagnostica Stago
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