Do ADP-activated platelets in vitro aggregate as discocytes ?

Thromb Haemost 2007; 98: 73–76 Ernst F. Lüscher had posed some interesting questions in 2000 about the time flow of the first platelet reactions after activation: shape change (SC) or aggregation (1). Regarding in-vivo studies – commencing on the observations of Bizzozero in 1882 (2) – Lüscher deduced from the literature and from own investigations (3, 4) that shape change which precedes aggregation would be an in-vitro artefact. Indeed, a plethora of ultra-structural studies dealt with the examination of aggregated platelets starting with the investigations of Born et al. 1980 (5), and Born and many others used transmission electron microscopy (TEM) to support the view that the initial decrease in light transmission in platelet aggregometry was caused by platelet shape change.Yet, recent morphological findings could attribute aggregates of discoid platelets (designated discocytes) to the initial change in the aggregometer curve (6). More recently, a shear-dependent in-vitro aggregation of discoid platelets was demonstrated using differential interference contrast microscopy or scanning electron microscopy (7–9). These initially unstable aggregates arise under the formation of membrane tethers between platelets and an adhesive surface. Tether formation involves the adhesive function of glycoprotein Ib/V/IX complex on von Willebrand factor.The conversion of aggregates from discoid platelets into stable aggregates consisting of platelets showing shape change requires the release of ADP. Apart from a study investigating aggregation in a genetic defective mouse (10), until now no in-vitro aggregate formation between discoid mammalian platelets could be observed in the TEM. Inspired by the aforementioned implications (1) we investigated whether it is possible to demonstrate aggregation of discoid platelets in vitro. To induce aggregation we chose ADP, an agonist of platelets released from damaged cells, particularly erythrocytes, at sites of vascular injury (11). Lüscher initiated these investigations and inspiringly accompanied them up to his sudden death in April 2002. Results presented in this report indicate that already 1.5 seconds (s) after ADP-stimulation of discoid platelets in-vitro fibrinogen molecules initiated the first contacts between platelets still retaining their discoid shape. The contacts were seen at any site of the platelet surface. One second later, shape change took place, and focal contacts were formed on the platelet body associated with the constricting contractile gel. The existence of bridging fibrinogen molecules during the aggregation was confirmed by immunolabelling with anti-fibrinogen antibody. Thus, platelet aggregation appears to start with discoid platelet-fibrinogen contacts under the chosen in-vitro conditions, and the twostage process leads to formation of stable aggregates of platelets with a changed shape.