Inhibition of coagulation in blood and blood products

Chemistry: analytical and immunological testing – Clotting or clotting factor level tests

Reexamination Certificate

Rate now

  [ 0.00 ] – not rated yet Voters 0   Comments 0

Details

C436S063000, C435S013000, C073S064410, C600S369000

Reexamination Certificate

active

06403381

ABSTRACT:

FIELD OF THE INVENTION
The present invention generally relates to methods for measuring coagulation of blood and blood products, and more particularly, to the use of an inhibitor to minimize blood product coagulation in vitro, and obviate sources of inconsistency and errors in blood coagulation. The present invention has a variety of uses, e.g., prolonging plasma clotting times, optimizing sensitivity of plasma coagulation assays, and enhancing storage of blood and blood products such as plasma.
BACKGROUND OF THE INVENTION
Blood coagulation (clotting) assists homeostasis by minimizing blood loss. In vivo, clotting usually requires vessel damage, platelet aggregation, coagulation factors and inhibition of fibrinolysis. The coagulation factors have been reported to act through a cascade that relates vessel damage to formation of a blood clot. See generally L. Stryer,
Biochemistry,
3rd Ed, W. H. Freeman Co., New York; A. G. Gilman et al.,
The Pharmacological Basis of Therapeutics,
9th Edition, McGraw Hill Inc., New York, pp. 1341-1359; and Mann, K. G. et al. (1992)
Semin. Hematol.
29: 213.
Initiation of blood coagulation arises from two distinct pathways: the intrinsic (contact) and extrinsic pathways. The intrinsic pathway can be triggered in vitro by contact of blood borne factor with artificial negatively charged surfaces such as glass. In contrast, the extrinsic pathway can be initiated in vivo or in vitro when tissue factor (TF) on a phospholipid surface, normally sequestered from the circulatory system, comes into contact with blood following injury. Both pathways are characterized by the assembly of multiple protein complexes on procoagulant surfaces, which serves to localize the response to the site of injury. See e.g, Mann, K. G. et al. (1990)
Blood
76: 1; Mann, K. G. et al. (1992), supra.
Current theories of coagulation maintain that interplay between the two pathways is required for efficient blood clotting. See S. I., Rapaport and L. V. M. Rao (1995)
Throm. Haemost.
74: 7; and Stryer, L. supra and references cited therein.
The contact pathway has been further divided into early and late steps. These steps are typically associated with specific coagulation factors. For example, the early contact pathway is associated with activated prekallikrein and Factor XII, whereas the late contact pathway involves Factors VIII and IX. It has been reported that hemophilia A, B and C are each correlated with deficiencies in the late contact pathway (Factor VIII, Factor IX, and Factor XI, respectively). Hemorrhagic tendencies have not been found for prekallikrein or factor XII deficiency. Accordingly, these early contact factors are not thought to be relevant for initiation and maintenance of coagulation. See e.g., Davie, E. W. et al. (1991)
Biochem.
30:10363.
Many activities of the extrinsic and intrinsic tenases (factor VIIIa-factor IXa) and the prothrombinase complex are facilitated by activated platelets and other phospholipid membranes. Characterization of the impact of therapeutic agents in vivo is usually analyzed by methods in which the contact pathway is attenuated or eliminated. See Nemerson, Y. (1988)
Blood
71:1, Rand, M. D. et al. (1996)
Blood
88: 1; and Monroe, D. M. et al. (1994)
Brit. J. of Haemot.
88: 364.
There have been attempts to understand how blood coagulation is initiated and controlled. One approach has been to reproduce blood coagulation as it is thought to occur in vivo. For example, by analyzing reactions associated with blood coagulation in vitro, it has been possible to detect relationships between certain blood coagulation factors. Specific attempts have involved analysis of fractionated blood and particularly blood products such as plasma. Current in vitro models of blood clotting focus on the activity of specific blood factors. See e.g., Davie, E. W. et al. supra.
Confusion about the role of these pathways in coagulation has arisen from several difficulties such as in processing, storing, and studying blood and blood products. Untreated whole blood or blood products such as plasma typically coagulate within minutes. The clotting can be reduced or eliminated by addition of a calcium-chelating agent such as citrate. In particular, citrate has been reported to interfere with the assembly and function of prothrombinase and extrinsic and intrinsic tenases. Citrated blood can be stored in liquid form for a limited period of time (e.g., days to weeks), or can be manipulated to produce blood products such as blood cell isolates, platelet rich and platelet poor plasma. Plasmas that include citrate can be stored for extended periods (months to years) by freezing at temperatures below about −70° C. In most instances, the plasma is recalcified for use. However, recalcified plasma will typically clot spontaneously due to contact activation in most storage vessels, where contact activation can occur within about 2 to 4 minutes. As a result, most coagulation assays are usually performed on citrated plasma fractions that have been frozen for storage then thawed. However, such fractions cannot be recalcified until immediately prior to use.
Coagulation tests are often performed either on blood or blood products. In simple tests such as bleeding time tests, wounds are made in a patient and the time until clot formation is noted. Additionally, whole blood coagulation tests have been devised by drawing blood directly into a tube, then rocking or agitating until a clot is observed. Such tests are not very informative, as the sources of initiation are not well controlled and comparisons among patients are difficult. In clinical settings, citrated plasma isolates are the most widely used blood product for coagulation testing, due to prominence of the prothrombin time (PT) and activated partial thromboplastin time (aPTT) tests. The PT is the more convenient assay, and is performed by addition of a large quantity of thromboplastin to the citrated plasma, with subsequent initiation of the reaction by calcium addition. The time to clot formation is noted, which for most normal donors is typically about 10 to about 14 seconds. The aPTT test involves about a 3 to about 5 minute preincubation of the citrated plasma with a mixture of phospholipids and solids possessing negatively charged surfaces. The reaction is initiated by calcium addition, and the clot time for normal donors typically falls between 25 and 43 seconds. While well established in the clinical venue, neither assay is entirely suitable to mimic the physiological coagulation reaction in its entirety. See generally
Williams Hematology,
infra.
For example, while the PT measurement employs the physiologically relevant initiator TF and the assay is sensitive to Factors V, VII, X, and prothrombin (II), the concentration used is sufficiently high that the reaction is usually insensitive to deficiencies or abnormalities in coagulation Factors VIII or IX. Clotting occurs rapidly in normal individuals (about 10 to about 14 seconds), and errors in measurement on the order of seconds are a significant fraction of the total clot time. When the assay is used to monitor administration of anti-coagulants, the target range for prolongation of the clot time is between about 2.5 to about 3.5 times normal, or between about 25 and 49 seconds. There has been recognition that this time range is often too small to permit accurate analysis.
The aPTT assay is also associated with problems. For example, since initiation proceeds through the early contact pathway members, Factor VII is bypassed in this reaction. As a result, this assay in insensitive to deficiencies or abnormalities in this biologically important coagulation factor. For this reason, the aPTT is not typically considered suitable for monitoring anti-coagulation by coumadin or other warfarin derivatives which strongly affect the ability of Factor VIIa to serve as an initiator of the coagulation reaction.
Additionally, most aPTT assays use plasma and are not compatible with whole blood. Thus, a source of phospholipid must often be provided, and the contribu

LandOfFree

Say what you really think

Search LandOfFree.com for the USA inventors and patents. Rate them and share your experience with other people.

Rating

Inhibition of coagulation in blood and blood products does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Inhibition of coagulation in blood and blood products, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Inhibition of coagulation in blood and blood products will most certainly appreciate the feedback.

Rate now

     

Profile ID: LFUS-PAI-O-2929385

  Search
All data on this website is collected from public sources. Our data reflects the most accurate information available at the time of publication.