Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving blood clotting factor
Reexamination Certificate
2000-10-11
2003-01-07
Leary, Louise N. (Department: 1623)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving blood clotting factor
C436S016000, C436S018000
Reexamination Certificate
active
06503724
ABSTRACT:
BACKGROUND OF THE INVENTION
Blood coagulation is a complex system involving a large number of proteins that function in concert to yield hemostasis. The coagulation system is regulated by a series of proteins present in plasma and on the surface of cells. Under physiological conditions, pro- and anti-coagulant mechanisms are delicately balanced to provide hemostasis and coagulation. Disturbances in this balance result in either bleeding or thromboembolic disorders, and can be induced by medical conditions, congenital or acquired, the intake of drugs or vitamins. The most widely used tests to measure the coagulation status of a person are known as clotting assays. Clotting assays measure the clotting times of recalcified platelet-poor plasma. The prothrombin time (PT) test is performed by adding calcium and thromboplastin to citrated plasma. The term thromboplastin refers to a phospholipid-protein extract of tissues, usually lung, brain, or placenta, that contains both tissue factor and the phospholipid necessary to promote the activation of factor X by Factor VII. The standard thromboplastin is “The World Health Organization” (WHO) thromboplastin which is derived from human brain thromboplastin. PT test instruments are based upon measuring how the plasma viscosity changes over time after calcium and thromboplastin have been added to plasma. When plasma coagulates it turns from a solution to a gel which is more viscous. In the APTT test, decalcified plasma is incubated for a specified time with activating agents such as ellagic acid, silica, soy extract, or kaolin. Following incubation with the activating substance, calcium chloride is added to the plasma mixture for a clot to form. In the PTT test, plasma is incubated for 3 minutes with a reagent supplying procoagulant phospholipid and surface-active powder (e.g. micronized silica). Calcium is then added and the clotting time noted.
Although widely used, clotting assays present several drawbacks. First, clotting assays are by definition invasive, as they require some blood to be drawn from the patient. Second, they require that actual coagulation of the blood be performed in a vial, which involves delicate handling, accurate timing, and citration of plasma. Third, clotting assays depend on an additional product, thromboplastin, whose performance can be different from the reference WHO thromboplastin, requiring complicated International Normalization Ratio (INR) calculations to ensure accuracy. Finally, clotting tests are indirect tests, reflecting only indirectly fibrinopeptides and fibrinogen degradation products and other proteins related to the state of anticoagulation.
Direct measurement of prothrombin fragments in blood or serum for determining the coagulation status is known in the art (U.S. Pat. No. 5,071,954). However, these methods are invariably invasive since they require some blood to be drawn.
Measurements of at least some of the above fragments in urine may be found in various papers, for instance in A. Bezeaud and al, Thrombosis Research, 13, 3, p. 551-556 (September 1978); J. V. Sorensen and al, Thrombosis Research, 67, 4, p. 429-434 (August 1992); D. M. Weinstock and al., American J. Hematology, 57,3, p. 193-199 (March 1998).
SUMMARY OF THE INVENTION
The purpose of this invention is to use a non invasive diagnostic test technique to measure the level of blood anticoagulation induced by pharmaceuticals which prevent or reduce coagulation of blood as well as to measure and/or identify any natural or disease-induced blood disorders which effect the coagulation of blood. In addition, the invention can be used to measure the level of hypercoagulation induced by drugs, proteins and vitamins which augment coagulation of blood (procoagulant drugs). The invention is a non invasive test since it measures key substances present in saliva, filtered saliva, sputum, or the like, which are obtainable non invasively (hereunder referred to as “saliva”). The invention is usable in humans as well as in animals.
It must be pointed out at this stage that saliva and urine are very different in terms or biological roles and compositions, as well as in their enzymatic environments.
Examples of clinical applications in which the test could be used include all blood coagulation disorders, be they congenital, acquired, or drug-induced. One can mention congenital deficiencies of the intrinsic pathway of the coagulation system (hemophilia A and B, deficiencies of Fitzgerald factor and Fletcher factor), deficiencies in protein C and protein S, heparin and heparin-like therapy, Warfarin (Coumadin)-like therapy, acute thrombotic situations such as acute myocardial infarction or pulmonary embolism, Vitamin K deficiency or excess, hypofibrinogenemia, liver disease, disseminated intravascular coagulopathy, among others.
The invention produces a quantitative measurement of prothrombin fragment 1+2 (F1+2), prothrombin fragment 1 (F1), prothrombin fragment 2 (F2), fibrinopeptide A (FpA) and D-Dimers in saliva which is correlated with the coagulation time as expressed for example by prothrombin time (PT), INR, partial thromboplastin time (PTT) and activated partial thromboplastin time (APTT). Fragments F1+2, F1, F2, FpA and D-Dimers are preferably measured by means of immunoassay techniques, which measure a substance using the reaction of an antibody (immunoreagent) with an antigen (i.e. the protein to be measured). The quantification of the substance measured is determined by measuring how much of the antigen is bound to the antibody and how much of the antigen is not bound to the antibody. Enzyme-labeled, fluorescent-labeled, phosphorescent-labeled, radio-labeled, chemiluminescent-labeled and bioluminescent-labeled immunoassay techniques are for example usable to measure the concentrations of Fragments F1+2, F1, F2, FpA and D-Dimers. Capillary action, precipitation, turbidometric, diffusion, agglutination and electrophorefic immunoassay techniques can also be used in practicing the invention, as well as potentiometric, amperometric, piezoelectric and evanescent-wave immunosensors. In addition, any combination of the aforesaid assay techniques can also be employed to measure the concentrations of Fragments F1+2, F1, F2, FpA and D-Dimers in saliva.
A preferred application of this invention is to control the level of any pharmaceutical which results in anticoagulation of the blood by affecting the conversion of blood Factor X to Factor Xa, which is commonly termed activated Factor X. Factor X and Factor Xa, which are in the latter stages of the coagulation cascade best correlate with the anticoagulation effect (see
FIG. 1
for diagram of coagulation cascade).
Factor X is a glycoprotein of molecular weight 55,000 and composed of two polypeptide chains linked by one disulfide bond. The light and heavy chains have molecular weights of 16,000 and 38,000 daltons respectively. During the coagulation process Factor X is converted to Factor Xa by either factors IXa, and VIII or Factor VII and tissue Factor. Factor X can also be activated by other proteases such as trypsin. The activation of Factor X by each of these systems involves the cleavage of a single specific arginyl-isoleucine peptide bond in the heavy chain of Factor X. This gives rise to the formation of a glycoprotein of molecular weight 44,000 and a peptide with a molecular weight of 11,000.
A common feature of Fragments F1+2, F1, F2, FpA and D-Dimers as measured in the invention is that their concentration rises when the conversion of Factor X to Factor Xa increases, and decreases when the conversion of Factor X to Factor Xa is inhibited. The respective roles of Fragments F1+2, F1, F2, FpA and D-Dimers in coagulation is explained in the following paragraphs.
Activated blood factor X forms a -complex with activated blood factor V, phospholipid and calcium. The first step in the activation by the complex is the proteolytic cleavage of a fragment from the NH2-terminal end of prothrombin. This gives rise to what is termed intermediate II (an intermediate precurs
Blasius Jacob R.
De Nora Matteo
Armstrong Westerman & Hattori, LLP
Leary Louise N.
Link Enterprises Corporation
LandOfFree
Fibrinopeptides in saliva as indicator of blood coagulation... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Fibrinopeptides in saliva as indicator of blood coagulation..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Fibrinopeptides in saliva as indicator of blood coagulation... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3072635