P-selectin increases adhesion of neutrophils to fibrinogen. (A) Neutrophils were isolated from fresh human blood and added to 96-well tissue culture plates coated with fibrinogen. For the dose-response curve, neutrophils were incubated with indicated concentrations of P-selectin Ig chimera (P-sel Ig). (B) Neutrophils were incubated with human IgG (hIgG) or P-selectin Ig chimera (P-sel Ig), and added to wells coated without fibrinogen (control) or with fibrinogen. For antibody inhibition experiments, P-selectin Ig chimera (P-sel Ig), was preincubated with G1 F(ab′)2 (a leukocyte adhesion-blocking mAb to P-selectin) or PS1 F(ab′)2 (a leukocyte adhesion-nonblocking mAb to P-selectin) prior to addition of neutrophils. Alternatively, neutrophils were preincubated with IB4 (a leukocyte adhesion-blocking mAb to β2 subunit), 44a (a leukocyte adhesion-blocking mAb to αM subunit), or mouse IgG (mIgG), prior to addition to the fibrinogen-coated wells. After washing, the bound neutrophils were quantified by MPO activities. The numbers of bound neutrophils were calculated according to the standard curves of MPO activities measured using the known amounts of neutrophils. All results are expressed as the mean ± SD values of the adherent cells determined in triplicate measurements of more than 3 separate experiments.
