Both unactivated and activated platelets adhere to immobilized fibrinogen, whereas only activated platelets adhere to fibrinogen fragment ‘D98.’ Calcein-labeled washed platelets (2 × 105/µL; 50 µL) were tested either before (unactivated) or after activation with 10 µM TRAP for adhesion to fibrinogen or ‘D98’ immobilized in microtiter wells by coating at 10 µg/mL. Platelets were incubated for 1 hour at 22°C in the presence or absence of EDTA (10 mM), 10E5 (20 µg/mL), 7E3 (20 µg/mL), or tirofiban (1 µM), followed by washing of unbound platelets and analysis of calcein fluorescence. The adhesion of unactivated platelets to fibrinogen was defined as 100% adhesion, and all values were normalized to this value in each of 8 separate experiments. (A) EDTA, 10E5, 7E3, and tirofiban inhibited adhesion of unactivated platelets to fibrinogen by 73%, 92%, 94%, and 86%, respectively (P < .0001 for all). (B) Activated platelets adhered to immobilized fibrinogen as well or better than unactivated platelets; EDTA, 10E5, 7E3, and tirofiban inhibited adhesion by 75%, 70%, 63%, and 63%, respectively (P < .001 for all). (C) Unactivated platelets adhered poorly to ‘D98’ (80% less adhesion compared with unactivated platelet adhesion to fibrinogen), whereas activated platelets adhered much better (25% more adhesion compared with unactivated platelet adhesion to fibrinogen). Adhesion was inhibited by 10E5, 7E3, and tirofiban by 66%, 63%, and 57%, respectively (P < .01 for all). (D) Adhesion of unactivated platelets to ‘D98’ was increased by treating the platelets with 10 mM EDTA (P = .02). Data reported as mean ± standard deviation (SD).
