Modifications in accessibility of membrane glycoproteins, binding of specific ligands and coagulation factor V during the activation of platelets in blood emerging from bleeding time wounds

Dual color flow cytometric techniques were applied to micro-aliquots of who le blood obtained from bleeding time (BT) wounds. Modifications in platelet activation markers (P-selectin [CD62P]) and lysosomal related protein (LIM PS [CD63]), presence of membrane glycoproteins (GPlb [CD42b], GPllb-llla [C D41a], GDIV [CD36], binding of von Willebrand factor (VWF), fibrinogen (Fg) and factor V [FV]) were analyzed in normal donors (n = 10)and in a severe von Willebrand patient (type 3) von Willebrand disease [vWD]), Samples of b rood (20 ul) were sequentially removed from BT wound edges for up to 5 min and fixed with paraformaldehyde, Antigens were detected using the correspon ding tagged monoclonal antibodies (moAbs) and quantitative results were ref erred to those found on platelet samples obtained from Venous blood obtaine d from the same individuals. A progressive increase in % of platelets posit ive for activation dependent antigens (CD62 from 7 +/- 2 to 48 +/- 19% and CD63 from 9 +/- 1 to 44 +/- 8%; initial vs. 4 min) was observed. Accessibil ity of GPllb-Illa epitopes on platelets from BT wounds remained slightly ab ove levels observed in Venous blood platelets, despite a progressive increa se in the presence of platelets positive for Fgn. Binding of MoAb to GPIV i ncreased at late stages of BT. A moderate decrease in the binding of a moAb to GPIb was observed on platelets obtained at late stages of the BT (14 +/ - 9% and 20 +/- 6% at 4 and 5 min, respectively). This apparent decrease in GPlb epitopes paralleled an increased presence of platelets positive far v WF (26 +/- 12 and 38 +/- 15%). Binding of moAb to GPlb always remained abov e basal levels in platelets obtained from BTs performed in the patient with type 3 vWD. Rr levers on platelets coming from the BT persisted at backgro und levels in all the individuals and at all times studied. Am. J. Hematol, 60:260-267, 1999, (C) 1999 Wiley-Liss, Inc.