Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Patent
1993-12-10
1996-12-24
Spiegel, Carol A.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
435 792, 435 794, 435 13, 435 7021, 43524027, 436501, 436529, 436548, 436 69, 53038826, G01N 3386, G01N 33577, G01N 33543
Patent
active
055872913
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND OF THE INVENTION
FIELD OF THE INVENTION
The invention describes a method for the quantitative determination of plasmin-.alpha.2-antiplasmin complexes as well as the use of this method as a mean of determining changes in the fibrinolytic system.
Introduction:
.alpha.2-antiplasmin is the most important plasmin inhibitor. Its rapid reaction with plasmin results in the formation of an inactive complex composed of one molecule of each component. Two steps are involved in this process: first, a reversible complex is formed between the lysine-binding site of plasmin and complementary sites on the carboxy terminal end of the .alpha.2-antiplasmin molecule. In a second step, an irreversible complex is generated associated with the cleavage of a peptide-bond in the inhibitor. During activation of plasminogen to plasmin an equilibrium exists between formation of plasmin-.alpha.2-antiplasmin complexes, occurring preferentially in the fluid phase and binding and action of plasmin on the fibrin surface. Bound to fibrin, plasmin is protected against inhibition by .alpha.2-antiplasmin. However, whenever fibrin is completely dissolved the plasmin liberated from the fibrin surface is immediately complexed by .alpha.2-antiplasmin.
Whenever fibrin forms in circulation, this process will be accompanied by activation of the fibrinolytic system because of the well known effects of fibrin on tissue plasminogen activator (t-PA). Plasmin generated thereby will in part become complexed with .alpha.2-antiplasmin leading to increased levels of plasmin-.alpha.2-antiplasmin complexes. Such increased levels of PAP-complexes have therefore been found in many circumstances in which fibrin formation is increased as in thrombophilia, hypercoagulability, disseminated intravascular coagulation, endotoxin shock, leukemia, liver diseases, nephrotic syndrome or after major surgery. Even in plasma after venous occlusion in most cases increased plasmin-.alpha.2-antiplasmin levels have been found consistent with increased fibrin formation and increased levels of tissue plasminogen activator in the venous occlusion plasma.
It is to be expected that during formation of serum a time dependent generation of PAP complexes occurs. The amount of PAP complexes generated in serum can be expected to be dependent on the amount of other plasminogen activators present in the respective environement as well as on plasminogen activator inhibitors, receptors and binding proteins.
While in all cases mentioned above only a limited increase of PAP levels is observed, thrombolytic therapy leads to extensive activation of the fibrinolytic system to a massive plasmin formation in the fluid phase and maximal PAP complex formation. Especially nonfibrin specific plasminogen activators such as streptokinase or urokinase can cause complete consumption of plasmin inhibitors resulting in an increased bleeding tendency due to plasminemia. Therefore the plasmin activity is no longer restricted to its specific substrate fibrin but can extend to nonspecific substrates as fibrinogen and other coagulation factors. Therefore, determination of plasmin-.alpha.2-antiplasmin complexes on the one hand can serve as indicative for general plasminemia during hyperfibrinolytic states with fibrinogen and .alpha.2-antiplasmin consumption and possible bleeding tendency; on the other hand, slightly increased levels of plasmin-.alpha.2-antiplasmin complexes are indicative for ongoing thrombus formation and thrombus dissolution as in the case of thrombophilia and PAP complexes in serum, respectively and are dependent on the total fibrinolytic potential.
Methods Available for Determination of PAP:
To determine PAP complexes several test methods have been published including two dimensional immunelectrophoresis, latex assay, RIA and more recently ELISA systems. Initially a latex agglutination assay for determination of PAP complexes was introduced by Plow et al. with rather low sensitivity. Then a two dimensional electrophoresis was described using the different mobility of free and comp
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Berbi Gesellschaft m.b.H.
Dubno Herbert
Myers Jonathan
Spiegel Carol A.
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