NEUTROPHIL CATHEPSIN-G MODULATES PLATELET P-SELECTIN EXPRESSION AND INHIBITS P-SELECTIN-MEDIATED PLATELET-NEUTROPHIL ADHESION

1. Close contact between platelets and neutrophils modulates their cel lular interactions in thrombotic and inflammatory states, with stimula tion of P-selectin expression on platelets by agonists such as thrombi n and neutrophil-derived cathepsin G being critical in mediating plate let-neutrophil adhesion. This study compared the effects of thrombin a nd cathepsin G on platelet P-selectin expression and on P-selectin-med iated platelet-neutrophil adhesion. 2. Washed platelets and platelet-n eutrophil mixed cell suspensions (platelet/neutrophil ratio, 10:1) wer e incubated with either the supernatant of activated neutrophils, puri fied cathepsin G or thrombin. Platelet P-selectin expression and plate let adhesion to neutrophils was quantified by flow fluorocytometric an alysis. 3. The supernatant from activated neutrophils stimulated plate let P-selectin expression comparable to that produced by purified cath epsin G or thrombin. P-selectin expression induced by both activated n eutrophil supernatant and purified cathepsin G was completely inhibite d by alpha(1)-antichymotrypsin, a specific inhibitor of cathepsin G, U nlike thrombin, which induced maximum platelet P-selectin expression b y 10 min, sustained to 120 min, cathepsin G induced an initial large i ncrease in platelet P-selectin expression, followed by a progressive r eduction over 30-60 min to baseline levels. 4. Co-incubation of neutro phils with thrombin-stimulated platelets resulted in a significant inc rease in P-selectin-mediated platelet-neutrophil adhesion, which was c ompletely inhibited by preincubation of neutrophils with anti-sialyl L ewis(x) monoclonal antibody. Thrombin produced maximum platelet-neutro phil adhesion by 10 min which remained stable over 120 min. In contras t, cathepsin G-stimulated platelets did not adhere to neutrophils over 120 min of co-incubation, Addition of cathepsin G to thrombin-stimula ted platelets caused a progressive reduction over 30-60 min to baselin e levels of platelet-neutrophil adhesion. 5. Neutrophil-derived cathep sin G is a potent platelet activator, but unlike thrombin it causes a time-dependent loss of platelet P-selectin expression and inhibits P-s electin-mediated platelet-neutrophil adhesion. Therefore, cathepsin G may modulate thrombin-mediated platelet-neutrophil adhesive interactio ns in inflammation and thrombosis.