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Abstract
Platelet function is influenced by the platelet thiol-disulfide balance. Platelet activation resulted in 440% increase in surface protein thiol groups. Two proteins that presented free thiol(s) on the activated platelet surface were protein-disulfide isomerase (PDI) and glycoprotein 1balpha (GP1balpha). PDI contains two active site dithiols/disulfides. The active sites of 26% of the PDI on resting platelets was in the dithiol form, compared with 81% in the dithiol form on activated platelets. Similarly, GP1balpha presented one or more free thiols on the activated platelet surface but not on resting platelets. Anti-PDI antibodies increased the dissociation constant for binding of vWF to platelets by approximately 50% and PDI and GP1balpha were sufficiently close on the platelet surface to allow fluorescence resonance energy transfer between chromophores attached to PDI and GP1balpha. Incubation of resting platelets with anti-PDI antibodies followed by activation with thrombin enhanced labeling and binding of monoclonal antibodies to the N-terminal region of GP1balpha on the activated platelet surface. These observations indicated that platelet activation triggered reduction of the active site disulfides of PDI and a conformational change in GP1balpha that resulted in exposure of a free thiol(s).
Highlights
Platelet function is influenced by the platelet thioldisulfide balance
Protein-disulfide isomerase (PDI)1 is a noncovalent homodimer with a subunit molecular mass of 57 kDa that catalyzes thiol-disulfide interchanges that can result in formation, reduction, or rearrangement of protein disulfide bonds
protein-disulfide isomerase (PDI) and GP1b␣ on the Platelet Plasma Membrane let surface. These findings demonstrated that platelet activation/aggregation triggered reduction of the active site disulfides of PDI and a conformational change in GP1b␣ that resulted in exposure of a free thiol(s)
Summary
Reagents and Chemicals—HEPES, apyrase (grade VII), leupeptin, phenylmethylsulfonyl fluoride, streptavidin-agarose, iodoacetamide, GSH, and dithiothreitol were purchased from Sigma, and Trasylol (aprotinin) from Bayer Australia, Sydney, New South Wales, Australia. The platelets were washed three times with PBS, lysed in 10 mM Tris-HCl, 0.15 M NaCl, pH 8.0, buffer containing 0.5% Triton X-100, 0.05% Tween 20, 10 M leupeptin, 10 M aprotinin, 2 mM phenylmethylsulfonyl fluoride, and 5 mM EDTA, sonicated as described above, clarified by centrifugation at 12,000 ϫ g for 30 min at 4 °C, and incubated with sheep anti-mouse coated Dynabeads (Dynal, Victoria, Australia) for 2 h at 4 °C on a rotating wheel. Binding of Anti-GP1b-IX Monoclonal Antibodies to Platelets—Resting platelets (2 ϫ 106 in 100 l of BSA-free Tyrodes buffer) were incubated with 200 g/ml of either preimmune rabbit IgG F(ab) fragments or rabbit anti-PDI F(ab) fragments for 30 min at room temperature. The samples were diluted 4-fold with BSA-free Tyrodes and kept in the dark until analysis
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