Effect of neutrophil granule secretion and platelet activation on levels of PSGL-1 and P-selectin in lysates of platelet-neutrophil suspensions.

(A) Purified and washed human platelets and neutrophils were resuspended in Tyrode buffer, pH 7.4, and were mixed together in a ratio of approximately 50:1 in Tyrode buffer alone or containing 5 μg/mL cytochalasin B, 0.1 μM fMLP, or 62.5 μM TRAP as shown. Some incubations were preincubated with a proteinase inhibitor cocktail that included 0.1 mM elastase inhibitor and chymostatin. After incubation at 22°C for 15 minutes, platelet-neutrophil suspensions were pelleted and treated with the proteinase inhibitor cocktail containing 1% Triton X-100. Aliquots of lysates were eluted on 7.5% SDS–polyacrylamide gels, transferred to membranes, and subjected to Western blot analysis using monoclonal antibodies against either P-selectin (AK4) or PSGL-1 (PL-1). The arrow indicates the expected position of the P-selectin fragment recognized by AK4 if P-selectin had been digested by cathepsin G. Data are representative of at least 3 experiments with different donors. (B) Purified¹²⁵I-labeled P-selectin was treated for 1 hour with purified human elastase or cathepsin G, eluted on 7.5% SDS–polyacrylamide gels, transferred to membranes, and subjected to Western blot analysis using monoclonal antibody against P-selectin (AK4).