A New Enzyme Immunoassay for Activation of Platelets by Biomaterials: Reduced Activation by Phosphorylcholine-Coated Sur
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A NEW ENZYME IMMUNOASSAY FOR ACTIVATION OF PLATELETS BY BIOMATERIALS: REDUCED ACTIVATION BY PHOSPHORYLCHOLINE-COATED SURFACES
EWAN J. CAMPBELL, MARTIN C. WILES, ROGER R.C. NEW & STEPHEN A. CHARLES, Biocompatibles Limited, Brunel Science Park, Kingston Lane, Uxbridge, Middlesex, UB8 3PQ, United Kingdom.
ABSTRACT. An enzyme immunoassay has been developed to semi-quantitate the degree of activation of platelets adhering to biomaterials. The method employs a monoclonal mouse anti-GMP-140 antibody. The glycoprotein GMP-140 is contained in alpha granules of resting platelets and is translocated to the outer membrane of platelets during activation via the thrombin-dependent pathway. A polyclonal anti-mouse IgG peroxidase conjugate is then added and platelet activation can be semi-quantitated by use of a suitable substrate, such as OPD. The results obtained by this method compare well with scanning electron microscopy, and are shown to be less prone to the misinterpretation often associated with other conventional assay systems such as the determination of platelet adhesion by detection of ATP. The adhesion and activation of platelets by biomaterials has been compared before and after coating these materials with phosphorylcholine (PC) derivatives. PC, in its form as a headgroup in membrane phosphoglyceride (lecithin) and ceramide (sphingomyelin) is a ubiquitous component of cell membranes, and displays extremely low interaction and binding with plasma proteins such as Factor XII [1,2L, complement [21, fibrinogen [3,2L, immunoglobulins [2] and albumin [2,4]. Using the immunoassay described here activation of platelets is seen to be dramatically reduced after coating surfaces, such as polyethylene, with PC compared with the untreated control. A PCcoated surface should therefore be an ideal starting point for developing tissue-inducing biomaterials. In addition endothelial cells contain GMP-140 in their secretory granules [5]. This would make this assay a potential tool in the investigation of endothelial cell - biomaterial interaction. Key Words: Haemocompatibility,Platelet Activation, GMP-140, Di-Acetylenic Phosphorylcholine.
Introduction. The interaction of platelets with biomaterials is thought to involve three stages:- platelet adhesion via the GPI~b/IIIa receptor, para-adhesion via the GPIb receptor [6] and activation resulting in shape change and degranulation. The GPIIb/IIIa receptor undergoes a conformational change upon activation of platelets to allow the binding of cellular matrix proteins, such as fibrinogen. Platelets may then adhere to fibrinogen adsorbed onto the biomaterial and become activated. In this process otgranules, dense-bodies and lysosomes migrate to the platelet surface where they fuse with the inner membrane surface thereby releasing their contents [7]. During degranulation, markers of platelet activation such as thromboxane A2, platelet factor 4 (PF4) and 0-thromboglobulin are released. In addition, neo-antigens, derived from the inner membrane of a-granules are expressed on the platelet surf
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