Approximately 6% of dog platelets are positive for staining with thiazole orange, a dye frequently used to stain ribonucleic acid. In this report, thiazole-orange positivity is shown to mark platelets that are less than 24 hours old. Dog platelets were derivatized in vivo with N-hydroxysuccinimido biotin such that greater than 95% of all platelets were biotinylated. Newly synthesized, nonbiotinylated platelets were then monitored by flow cytometry for their ability to bind thiazole orange. After biotinylation, the percentage of biotin-negative, thiazole-orange-positive platelets increased gradually from 0.72% at 30 minutes to 5.44% at 24 hours. These data indicate that thiazole-orange staining does label newly synthesized platelets.
Dog and human fibrinogen were derivatized with N-hydroxysuccinimidofluorescein and utilized for flow cytometric estimation of fibrinogen binding to activated platelets. Fluorescein-fibrinogen binding fulfilled the criteria for specific binding to platelets; the binding was saturable, dependent on agonist activation, and inhibited by unlabeled fibrinogen. In addition, EDTA and barbourin, a KGD-containing peptide, were found to inhibit the binding of fluorescein-fibrinogen. Fluorescein-fibrinogen bound to dog platelets with an apparent affinity of 0.31 p M after stimulation with either adenosine-5'-diphosphate (ADP) or plateletactivating factor. The labeled fibrinogen was also used to study the fibrinogen binding capacity of aged, biotinylated platelets. Aged platelets were indistinguishable from young platelets with regard to fibrinogen binding in response to ADP. These studies document that direct derivatization of fibrinogen with fluorescein generates a useful probe for analyzing fibrinogen binding to platelets with flow CytOmetry. 0 1994 Wiley-Liss, Inc.Key terms: Protein labeling, fluorescent tag, activated platelets, N-hydroxysuccinimido-fluorescein Fibrinogen (Fbg) is a multimeric, 340 kDa plasma protein required for platelet aggregation and clot formation; the critical nature of the fibrinogenlplatelet interaction is indicated by the bleeding tendency of patients deficient in platelet receptors responsible for fibrinogen binding (for review, see 18). The interaction of Fbg with activated platelets occurs primarily through the integrin receptor complex, glycoprotein (GP) IIb-IIIa (cqIbP3; 19,241. Two different classes of sites within the Fbg molecule have been reported to be involved in its recognition by GP IIb-IIIa. Kloczewiak et al. (13) identified the carboxy-terminal sequence on the gamma chain, y406-411, as the minimum sequence mediating Fbg binding to platelets. In addition, the Aa-chain has two RGD sequences, a recognized ligand for the integrin receptor family (1 1). The gamma chain and RGD sequences of Fbg appear to share a common binding site on glycoprotein , and synthetic peptides corresponding to these sequences can inhibit Fbg binding to platelets (27) and platelet aggregation (6). Recent observations suggest that the gamma chain dodecapeptide is responsible for the initial interaction of Fbg with activated GP IIb-IIIa (7) and that the RGD sequences might be more specifically involved in postreceptor occupancy events (26,301.The binding of Fbg to activated platelets has traditionally been analyzed with radiolabeled Fbg. Such studies document that approximately 50,000 binding sites are present on fully activated human platelets with a n apparent affinity of 0.18 pM and that this binding is totally inhibited by EDTA (25). In addition, there are several reports in the literature utilizing fluorescein isothiocyanate (FITCI-labeled anti-Fbg antibodies to detect bound fibrinogen on activated platelets by flow cytometry (FCM). These techniques have not enjoyed widespread acceptance and are complicat...
The alpha granules of circulating platelets are dynamic structures that acquire endogenous and exogenous components by synthesis and uptake, respectively. The uptake of exogenous components is a result of either receptor-mediated endocytosis or fluid-phase pinocytosis. Despite many detailed studies on the function and content of alpha-granules, little is known of the impact of platelet age on these organelles. In this report, we describe the use of platelet biotinylation to identify and isolate aged platelets for the analysis of alpha-granule contents. When aged platelets were permeabilized and examined by flow cytometry utilizing fluorescently labeled antibodies, two exogenously acquired proteins, fibrinogen and immunoglobulin G, were found to increase significantly with platelet age. The levels of intracellular fibrinogen were found to be elevated relative to control, 114 +/- 2% and 119 +/- 5% on days 4 and 5 postbiotinylation, respectively; the life span of dog platelets is 6.0 days. Intracellular immunoglobulin G content increased similarly. Levels of two endogenously synthesized proteins, thrombospondin and P-selectin, were not elevated in aged platelets. Confirmation of the flow cytometric data was obtained by isolating aged, biotinylated platelets by fluorescence-activated cell sorting and quantitating the fibrinogen levels with an ELISA assay. For platelets averaging 4.6 days of age, the fibrinogen level was elevated to 128 +/- 23% of the level for the entire platelet population. These data demonstrate that age-dependent changes in exogenously acquired alpha-granule proteins do occur and that the uptake mechanism for these proteins is active throughout the platelet life span.
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