Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving blood clotting factor
Reexamination Certificate
1997-09-05
2001-03-27
Saunders, David (Department: 1644)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving blood clotting factor
C436S069000, C530S300000, C530S330000
Reexamination Certificate
active
06207399
ABSTRACT:
BACKGROUND OF THE INVENTION
The subject invention lies in the field of thrombosis and haemostasis. In particular the invention is directed at improved chromogenic substrates for thrombin. The substrates according to the invention can be used to great advantage in determination of endogenous thrombin potential. The determination of endogenous thrombin potential and advantages and applications thereof are described in EP appl.nr. 902025097, published on Apr. 3, 1991, publication number 420332 of Hemker et al. The content of said patent is hereby incorporated by reference. The subject invention is also directed at improved methods of ETP determination in a continuous assay.
INTRODUCTION
Thrombin Generation: Its Assessment Via the Endogenous Thrombin Potential
Thrombin is a pivotal element in the complex interplay of vessel wall, blood cells and plasma proteins that ensures haemostasis but also may lead to thrombosis. The mechanisms of prothrombin activation and thrombin inactivation are closely intertwined with functions of the formed elements of blood and blood vessels. The convenient oversimplification of older text books no longer hold: Primary haemostasis and arterial thrombosis are no longer thought to be uniquely platelet functions; secondary haemostasis and venous thrombosis are not the in vivo equivalents of blood coagulation in a tube. Thrombin is the most powerful platelet activator and platelet activation is a prerequisite for normal thrombin formation. The vessel wall carries important parts of the thrombin generation mechanism and periluminal cells carry the tissue factor, the initiator of thrombin generation. The importance of thrombin generation is well illustrated by the fact that all pharmaceutical interventions that decrease thrombin generation (O.A.C., pentasaccharide) or that increase thrombin breakdown (classical heparin, dermatan sulphate) have an antithrombotic action and upon overdosage, cause bleeding. On the other hand all congenital conditions that increase thrombin generation (deficiencies of proteins C and S. APC resistance) or that decrease thrombin breakdown (deficiency of AT III) will cause a thrombotic tendency.
In view of the central role of thrombin it is important to be able to assess the thrombin generating capacity of a plasma sample in a single, global function test; i.e. express the resultant of the thrombin-forming and thrombin-breakdown mechanisms in one parameter. This figure would show a decrease upon hypocoagulation of any kind and an increase in hypercoagulability. For more than a century clotting times have been used for this purpose, but they are not sensitive to hypercoagulation, not very sensitive to moderate coagulation defects and sometimes insensitive to anticoagulant measures (PT to heparin: PT and APTT to low molecular weight heparins). This is mainly because clotting is an early event during the process of thrombin generation. At the moment a plasma sample clots, the large majority (>95%) of thrombin still has to be generated and variations in this process are not reflected in the clotting time. In the APTT the lag time of thrombin formation is mainly dependent upon the feedback activation of factor VIII, which in itself is a function of thrombin generation, which is why this test is the best one available at this moment to measure e.g. the heparin effect.
As a parameter that reflects the whole of the thrombin generation process the Endogenous Thrombin Potential (ETP) i.e. the area under the thrombin generation curve has been proposed. ETP is indeed an indicator of the potency of the clotting mechanism.
The potency of thrombin generation should not be confounded with the extent of ongoing thrombin generation in the body. Fragments 1, 2 of prothrombin and TAT-complexes reflect how much thrombin is generated in the body and subsequently inactivated. They are like smoke detectors reporting an ongoing fire. A test for hypo- of hypercoagulability however should indicate the potential capacities of the non-triggered system. In D.I.C. with consumption coagulopathy the indicators of ongoing coagulation are high but the capacity of the plasma to generate thrombin is low. In congenital AT III deficiency the reverse is the case.
The Mechanism of Thrombin Generation
The mechanism of thrombin generation is governed by three different kind of processes, the three “axes” of thrombin formation:
a) Thrombin production and inactivation in the strict sense of the word,
b) Modulations of the thrombin generation velocity and
c) Localisation at the site of vascular damage.
These three axes are best illustrated in the prothrombinase complex. The production of thrombin from prothrombin is caused by factor Xa, the availability of factor Va determines the velocity of thrombin generation and the process is localised at the surface of a procoagulant membrane.
The core of the thrombin generation mechanism is the production-inactivation axis: Tissue factor activates factor X, which activates prothrombin. The resulting thrombin is inactivated by antithrombin and minor inhibitors. The production along the axis is limited in time by the TFPI mechanism. When a sufficient amount of factor Xa is formed, the Xa generation is shut down because of formation of Xa-TFPI complexes that efficiently inhibits the factor VIIa-tissue factor complexes. To prevent precocious arrest of thrombin formation via the TFPI mechanism, the Josso Loop constitutes an escape mechanism: Factor IX is activated by the factor VIIa-TF complex like factor X is. Together with factor VIIIa it forms an alternative factor X activator that is not inhibited by TFPI but the activity of which is modulated by factor VIII activation and inactivation, a process entirely comparable to the modulation of prothrombinase activity by factor V(a). The second axis is modulation of thrombin generation via factor V activator and inactivation. Factor Va enhances the turnover of prothrombin by factor Xa some thousand fold. The appearance and disappearance of factor Xa is governed by thrombin. Factor Xa has been shown to activate factor V in purified systems but not in plasma. The natural activator of factor V in a tissue factor activated system is probably meizothrombin. This can i.a. be deduced from the fact that the thromboplastin time is relatively insensitive to heparin, meizothrombin being insensitive to AT III—heparin action. The inactivation of factor V is indirectly thrombin dependent because the scavenger of factor Va, activated protein C, is generated by the thrombomodulin-thrombin complex.
Factor VIII governs factor IXa dependent generation of factor Xa in the same way factor V governs prothrombinase activity. A difference is that factor V is probably activated by meizothrombin at a phospholipid surface whereas factor VIII is kept in solution by von Willebrand factor and is activated by thrombin.
The third axis of thrombin generation is localisation. Upon loss of endothelial integrity, platelets adhere to the subendothelial material. Cell damage exposes first traces of procoagulant (PS containing) membranes and Tissue Factor starts the coagulation cascade. As soon as traces of thrombin are formed, the simultaneous action of thrombin and collagen induces transbilayer movement of PS in the platelet membrane and the platelet surface becomes procoagulant. The procoagulant action of PS containing membranes is due to the fact that clotting factors adsorb to these surfaces. This increases the effective concentration of the reactants. It has been shown e.g. that prothrombin adsorbs at such surfaces and then, by diffusion in the plane of the surface, reaches the prothrombinase complex. Because the chance for prothrombinase and prothrombin to meet is much bigger in two dimensions than in three, this mechanism “guides” prothrombin to prothrombinase. The extent of the procoagulant surface around a prothrombinase molecule has indeed been shown to determine the apparent Km of prothrombin conversion, i.e. the concentration of prothrombin in solution necessary to half-saturate prothrombinase.
The whole of thrombin
Hemker Hendrik Coenraad
Rijkers Dirk Thomas Sigurd
Tesser Godefridus Ignatius
Hemker Hendrik Coenraad
Pillsbury & Winthrop LLP
Saunders David
Tung Mary Beth
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