Gold labeling of thrombin and ultrastructural studies of thrombin-gold conjugate binding by fibrin |
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Authors: | C Y Liu D A Handley S Chien |
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Affiliation: | 1. Dokuz Eylul University, Graduate School of Natural and Applied Science, Tınaztepe Campus, 35160, Izmir, Turkey;2. Dokuz Eylul University, Chemistry Department, Tınaztepe Campus, 35390, Izmir, Turkey;1. USDA/ARS National Coldwater Marine Aquaculture Center, Franklin, ME 04634, USA;2. USDA/ARS Warmwater Aquaculture Research Unit, Thad Cochran National Warmwater Aquaculture Center, PO Box 38, Stoneville, MS 38776, USA;3. Aquaculture Research Institute, Department of Fish and Wildlife Sciences, University of Idaho, Hagerman, ID, USA |
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Abstract: | Monodispersed thrombin-gold (T-Au) conjugates were prepared by the absorption of a monolayer (3.8 nm thick) of human alpha-thrombin around individual monodispersed colloidal gold particles (16.5 +/- 1.8 nm). Like free molecular thrombin, T-Au conjugates can cause platelet aggregation, plasma clotting, and the release of fibrinopeptides A and B from fibrinogen. At the same thrombin concentration, T-Au conjugates have only one-tenth the fibrinogen-clotting activity of free thrombin and one-third the amidolytic activity of free thrombin. Hirudin can completely inhibit the fibrinogen-clotting activity of both T-Au conjugates and free thrombin, but can inhibit only half of the amidolytic activity of the conjugates. Diisopropyl fluorophosphonate can completely inhibit the fibrinogen-clotting activity and the amidolytic activity of both T-Au conjugates and free thrombin. T-Au conjugates were further characterized by studying the mechanism of their binding to fibrin and the location of the binding site on fibrin. The results of electron microscopic studies showed that T-Au conjugates, but not albumin-Au conjugates, are bound by fibrin. Increasing T-Au conjugate concentrations are associated with an increase in the number of T-Au conjugates binding to fibrin. At 0.1 microM thrombin, 73% of the T-Au conjugates are bound to branch points of the fibrin network with 27% of the T-Au conjugates present in the fibrin strands. At higher thrombin concentration (e.g., 0.5 microM) the percentage of T-Au conjugates bound to locations other than branch points increases to 62%.(ABSTRACT TRUNCATED AT 250 WORDS) |
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