Fluorescein derivatization of fibrinogen for flow cytometric analysis of fibrinogen binding to platelets

Abstract

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...

Modulation of ADP-Induced Platelet Activation by Aspirin and Pravastatin: Role of Lectin-Like Oxidized Low-Density Lipoprotein Receptor-1, Nitric Oxide, Oxidative Stress, and Inside-Out Integrin Signaling

“…Then, 10 M ADP was added together with 200 g/ml Alexa Fluor 488-human fibrinogen, and the sample was incubated at 37°C for 15 min (Heilmann et al, 1994). Alexa Fluor 488-fibrinogen binding to platelets was determined by flow cytometry using FACSCalibur.…”

Evaluation Of Platelet Function By Flow Cytometry

“…Other αIIbβ3-specific activation-dependent monoclonal antibodies are directed against either ligand-induced conformational changes in αIIbβ3 (ligand-induced binding sites, LIBS) 18 or receptorinduced conformational changes in the bound ligand (fibrinogen) (receptor-induced binding sites, RIBS) 19 (Table 3). Rather than αIIbβ3-specific monoclonal antibodies, fluorescein-conjugated fibrinogen can also be used in flow cytometric assays to detect the activated form of platelet surface αIIbβ3, 20,21 but the concentration of unlabeled plasma fibrinogen and unlabeled fibrinogen released from platelet α granules must also be considered in these assays. The most widely studied type of activation-dependent monoclonal antibodies directed against granule membrane proteins are P-selectin (CD62P)-specific.…”

Flow cytometric measurement of kinetic and equilibrium binding parameters of arginine-glycine-aspartic acid ligands in binding to glycoprotein IIb/IIIa on platelets

“…Recently, the use of a flow cytometric method for measurement of equilibrium binding of fluorescein-labeled fibrinogen (11), disintegins (18), and RGD peptide (22) to GPIIb/IIIa has been demonstrated. In this paper, we describe the flow cytometric measurements of dynamic and equilibrium binding parameters of two new RGD peptidomimetics, L-762,745 (5) and L-769,434 (for scheme 1, see Fig.…”