Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Patent
1995-10-04
1998-09-22
Achutamurthy, Ponnathapura
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
435 4, 435 71, 435 72, 436518, G01N 3353, G01N 33543, G01N 3386
Patent
active
058112502
DESCRIPTION:
BRIEF SUMMARY
The present invention is concerned with the detection and/or measurement of microvesicles derived from blood platelets in a liquid sample such as plasma and the use of this measurement in the diagnosis of haemostatic disorders.
Blood platelets are a normal component of blood and are known to participate actively in haemostatic processes. Through their interaction with the subendothelium of damaged blood vessels, for example, platelets may aggregate to form a primary haemostatic plug. This process is also usually accompanied by the production of various factors involved in haemostasis by the platelets.
Activation of blood platelets greatly enhances their catalysation of haemostatic reactions, and in particular activated platelets demonstrate an increased concentration and activity of factor Va. (Factor Va is a component of the so-called prothrombinase complex which produces thrombin from its precursor prothrombin.) In addition it has been found that platelets shed microvesicles (also called microparticles) from their plasma membrane upon activation. This phenomenon was first described by Sandberg et al. (Biochem. J. 203: 303-311 (1982)).
Following further research, it is now believed that the platelet microvesicles are formed by pinching off or budding of the plasma membrane. The microvesicles contain the platelet glycoproteins Ib, IIb and IIIa and the cytoskeletal proteins filamin, talin and myosin. The microvesicles formed have a diameter of from 50 to 800nm.
Both the microvesicles and the platelets are able to catalyse haemostatic reactions and each display both procoagulant and anti-coagulant activities. Whilst it has been postulated that the formation of microvesicles is linked to production of the prothrombinase complex (see Sims et al., J. Biol. Chem. 264: 17049-17057 (1989)) it has not however been demonstrated that any specific activity is confined either to the platelet derived microvesicles or to the platelets themselves.
Platelet activation and microvesicle formation can be induced by a number of different factors, for example thrombin, collagen, ionophores, calpain and complement protein complexes eg. C5b-9. Weak activators of platelets, for example ADP, epinephrine and platelet activating factor cause only low levels of microvesicle formation. Increased concentration of microvesicles has been demonstrated following storage of platelet concentrates (Bode et al., Blood 77: 887-895 (1991); Solberg et al., Thromb. Res. 48: 559-565 (1987)). Additionally, it has also been shown that stirring of samples comprising platelets activated by collagen and/or thrombin will enhance microvesicle formation.
The formation of haemostasis in causing or reflecting the development of vascular disease or disorders is widely recognised and several haemostatic factors have been examined for their suitability as markers of such disease and disorders. Thus prothrombin time tests involving thromboplastin or activated partial thrombin time tests involving activators for intrinsic plasma coagulation may be used to monitor or to detect disease. Also used are tests for fibrinogen, antithrombin III, protein C, protein S, factor VIII, tissue plasminogen activator, fibrin monomers and fibrinogen degradation products.
As an alternative to the measurement of plasma haemostatic factors, attempts have also been made to monitor the degree of cellular activation taking place during haemostasis. The activation of platelets has been investigated in this regard since (as discussed above) platelets are important participants in haemostasis and are also believed to play a major role in the development of arterial thrombosis and in atherosclerotic processes.
Previous studies have endeavoured to monitor platelet activation by measuring the amounts of proteins secreted. Thus, assays for the measurement of platelet factor 4 (PF-4), .beta.-thromboglobulin (.beta.-TG) and thrombospondin have been investigated (see Lane et al. Thromb. Haemostas. (Stuttgart) 52(2) pages 183-187 (1984)). However, none of these proteins were found to be suitable fo
REFERENCES:
patent: 5266462 (1993-11-01), Hemker et al.
patent: 5627036 (1997-05-01), Reutelingsperger
George et al., The Journal of Clinical Investigation, vol. 78, No. 2, Aug. 1986, pp. 340-348.
Sandberg et al., The Biochemical Journal, vol. 203, 1982, pp. 303-311.
Sims et al., The Journal of Biological Chemistry, vol. 264, No. 29, 15 Oct. 1989, pp. 17049-17057.
Bode et al., Blood, vol. 77, No. 4, 15 Feb. 1991, pp. 887-895.
Brosstad Frank
Gogstad Geir Olay
Holme Pal Andre
Solum Nils Olay
Achutamurthy Ponnathapura
Nycomed Pharma A/S
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