Low-Voltage, High-Resolution, Scanning Electron Microscopy of Platelet-Bound Fibrinogen

Abstract

An essential element in blood clot formation is fibrinogen-mediated platelet aggregation. Fibrinogen is an adhesive plasma protein which binds to the αIIbp3 integrin on activated platelet surfaces. Platelets do not aggregate in the absence of fibrinogen binding, and fibrinogen bound to surfaces of platelets in aggregates is localized to regions of platelet-platelet contact. The fibrinogen molecule is symmetrical and bifunctional and may directly bridge the gap between platelets to bind to receptors on two adjacent platelets. However, the precise mechanism by which fibrinogen links platelets is unclear.Previously we have utilized colloidal gold labeling with correlative light and electron microscopy to investigate the binding of fibrinogen to receptors on surfaces of spread, substrate adherent platelets. The initial binding of gold-conjugated fibrinogen (FgnAu) and subsequent ligand-triggered receptor movement was followed on living platelets by video-enhanced light microscopy. Fibrinogen receptors initially are dispersed over much of the platelet surface and move centripetally upon fibrinogen binding, ultimately forming a band of bound fibrinogen on the platelet surface overlying a densely woven band of actin filaments surrounding the central granulomere. After preparation for electron microscopy, the same platelets as were followed in the light microscope were located in the high voltage TEM and the low voltage, high resolution, SEM (Hitachi S-900) and the final locations of the gold labeled receptor/ligand complexes were determined relative to internal or surface ultrastructure, respectively. More recently, we have utilized the SEM operated at low (1-2 kV) beam voltage to examine in detail the binding of unlabeled fibrinogen to platelets. With appropriate specimen preparation, individual cell surface macromolecules can be resolved in situ by low voltage SEM. In addition to the centripetal receptor redistribution seen with FgnAu, unlabeled fibrinogen appeared to undergo self-adhesive interactions following binding to platelet fibrinogen receptors, forming small, branched and globular protein aggregates during translocation across the platelet surface.(Fig. 1)